Cowan and Steel's Manual for the Identification of Medical Bacteria, Third Edition

This standard and internationally known reference manual for the identification of medically important bacteria, Cowan and Steel, occupies an essential place at the bench of all medical microbiologists. The material in this new edition, which follows the successful pattern of previous editions, has been extensively revised, and is suitable for use in all medical bacteriology laboratories using traditional diagnostic methods. The core of the manual is the series of diagnostic tables which, with the accompanying descriptive text and definitions, give the characteristics of all bacteria likely to be encountered in public health laboratories, and in medical and veterinary practice. This edition contains new sections on rapid and mechanized test methods and on the laboratory applications of computers to the identification of bacteria. The importance of laboratory quality control and proficiency procedures is emphasized throughout. The Appendices give details of laboratory methods and media for all the recommended diagnostic tests, and provide abstracts of the official guidelines for bacterial nomenclature. As in previous editions, the text contains comprehensive and up to date references.

Cowan and Steel's manual for the identification of medical bacteria

COWAN AND STEEL'S Manual for the identification of medical bacteria THIRD EDITION EDITED AND REVISED BY

G. I. BARROW M.D., F.R.C.Path., Dip. Bact. formerly Consultant Medical Microbiologist, Public Health Laboratory Service and Director, Public Health Laboratory, Truro, Cornwall and

R. K. A. FELTHAM B.Sc, Ph.D., M.I.Biol. formerly Principal Microbiologist and Director ACT Medisys Ltd, Edgbaston, Birmingham, and Senior Microbiologist, Public Health Laboratory, Leicester

CAMBRIDGE UNIVERSITY PRESS

PUBLISHED BY THE PRESS SYNDICATE OF THE UNIVERSITY OF CAMBRIDGE The Pitt Building, Trumpington Street, Cambridge, United Kingdom CAMBRIDGE UNIVERSITY PRESS The Edinburgh Building, Cambridge CB2 2RU, UK 40 West 20th Street, New York NY 10011-4211, USA 477 Williamstown Road, Port Melbourne, VIC 3207, Australia Ruiz de Alarcon 13, 28014 Madrid, Spain Dock House, The Waterfront, Cape Town 8001, South Africa http ://www. Cambridge. org © Cambridge University Press 1965, 1974, 1993 This book is in copyright. Subject to statutory exception and to the provisions of relevant collective licensing agreements, no reproduction of any part may take place without the written permission of Cambridge University Press. First published 1965 Second edition 1974 Third edition 1993 Reprinted 1995, 1999 First paperback edition 2003 A catalogue record for this book is available from the British Library Library of Congress Cataloguing in Publication data Cowan and Steel's manual for identification of medical bacteria 3rd ed. / edited and rev. by G.I. Barrow and R.K.A. Feltham. p. cm. Includes bibliographical references. Includes index. ISBN 0 521 32611 7 hardback 1. Diagnostic bacteriology - Handbooks, manuals, etc. I. Cowan, S.T. (Samuel Tertius) II. Steel, KJ. (Kenneth John) III. Barrow, G.I. IV Feltham, R.K.A. V Title: Manual for the identification of medical bacteria. [DNLM: 1. Bacteria — classification. QW 15 C874] QR67.2.C68 1993 616.014-dc20 90-2511 CIP ISBN 0 521 32611 7 hardback ISBN 0 521 54328 2 paperback

Contents

Foreword Preface to the first edition Postscript Preface to the second edition Preface to the third edition List of contributors Introduction 1 Classification and nomenclature 1.1 Classification 1.2 Nomenclature

ix xi xi xii xiii XV

xvii 1 1 4

2 Culture media: constituents and sterilization 2.1 Media for different purposes 2.2 Media constituents 2.3 Indicators 2.4 Sterilization

12 12

3 Principles of isolation 3.1 Isolation methods 3.2 Importance of pure cultures 3.3 Screening tests 3.4 Rapid tests

15 15 15 19 20

4 Bacterial characters and characterization 4.1 Bacterial characterization 4.2 Choice of characters 4.3 Characters not used in the tables 4.4 Primary tests used in the tables 4.5 Secondary tests used in the tables 4.6 Rapid methods for the screening and identification of bacteria

7 /

Q O

Q y

21 21 22 22 24 28 42

5 Theory and practice of bacterial identification 5.1 5.2

46 46 48

Theory of identification Practice of identification

6 Characters of Gram-positive bacteria 6.1 6.2 6.3 6.4 6.5 6.6

Division into major groups The staphylococci and micrococci The streptococci The anaerobic Gram-positive cocci The diphtheroids The coryneform-actinomycete intermediate group 6.7 The actinomycetes 6.8 The anaerobic bacilli 6.9 The aerobic spore-formers 6.10 The acid-fast rods

7 Characters of Gram-negative bacteria 7.1 7.2 7.3 7.4 7.5

Division into major groups The Gram-negative anaerobes The Gram-negative aerobic cocci The Moraxella-Acinetobacter group The non-(or weakly) saccharolytic motile rods 7.6 The actively saccharolytic motile rods 7.7 The Pasteurella and pasteurella-like group 7.8 The Vibrio and vibrio-like group 7.9 The enterobacteria 7.10 A group of difficult organisms

8 Taxonomy in theory and practice 8.1 8.2

Different kinds of classification Bacterial nomenclature and coding

Vll

50 50 52 59 68 69 75 79 83 86 90 94 94 94 98 102 107 109 117 121 128 150 165 165 168

CONTENTS

8.3 8.4

Bacterial identification Bacterial taxonomy in the future

171 173

9 Bacterial identification by cards 9.1 Minimal-difference Identicards for genera 9.2 Identicards for species

175 176

10 Bacterial identification by computer 10.1 Strain collections 10.2 Test selection 10.3 Identification of an unknown isolate 10.4 Identification: statistical principles 10.5 Profile numbers

179 179 180 180 181 182

11 Quality control in microbiology 11.1 Laboratory quality control 11.2 Laboratory proficiency and quality assessment 11.3 Laboratory safety and hygiene

184 184 186 187

Appendices A Preparation and control of culture media Al General considerations A2 Formulae of media A3 Media control

188 188 192 208

B Staining: reagents and methods B1 Reagents B2 Methods

214 214 216

Vlll

175

C Characterization tests Cl Reagents C2 Buffer solutions C3

Characterization test methods

219 219 222 222

D Test organisms

239

E Preparation and use of Identicards

243

F Use of computers

249

G The Bacteriological Code

252

H The Approved Lists of Bacterial Names

254

I Reconciliation of Approaches to Bacterial Systematics J Glossary Jl Taxonomic terms J2 Abbreviations and acronyms

260 263 263 266

References

268

Index

317

Foreword

For over 25 years now, medical bacteriologists all over the world have turned to 'Cowan and Steel' as their first reference book when they encountered an unfamiliar bacterial isolate. A generation of laboratory workers has grown up with it. They turned to it not only because there was clear information on how to examine isolates, with concise details of culture media and test methods that were applicable to the great majority of bacteria of medical importance, but also because of the famous successive tables that led from genera with their minidefinitions to species with their characters. These were combined with practical hints on where one might go wrong, and succinct information on the pathogenic species. The tables contained carefully chosen data in just the right amount for a useful laboratory manual on the identification of medically important bacteria. In the years since the last edition, test methods and the variety of bacteria of medical interest have both grown considerably. Not only have poorly studied areas like the 'diphtheroids' been much clarified, but a number of newly recognized pathogens such as

legionellae have become important. Medical and other workers will therefore welcome this new edition, which follows closely the emphasis and style of its predecessors. The contributors and editors are to be congratulated on their labours in bringing a complex field to the concise summary that is contained here, often in the face of difficulties in finding convenient diagnostic tests for the newer taxa. Some new features in this edition will greatly help users. The chapters on theory and practice in bacterial taxonomy, on computer identification and on bacterial nomenclature will be especially welcomed. The further emphasis on quality control and proficiency assessment procedures, both within laboratories and between them, will also be most useful. It is a pleasure to be able to recommend this Manual wholeheartedly to those concerned with medical and associated bacteriology everywhere. Leicester

P.HA. Sneath

IX

Preface to the first edition

Our 'Diagnostic Tables for the Common Medical Bacteria' were originally published in the Journal of Hygiene. The tables seemed to fill a need and the demand for reprints was so great that Cambridge University Press reprinted them in pamphlet form. Many inquired about the technical methods, and there were constant complaints that the methods were not described and that the text lacked details of the taxonomic problems. We resolved, therefore, to expand the original paper and to prepare a book which would give sufficient detail of media and methods to justify its description as a laboratory manual. Although designed for medical workers we hope that others will use it. The value of a laboratory manual was impressed on one of us in 1935 at the British Postgraduate Medical School. Dr A. A. Miles had prepared a loose-leaf mimeographed manual to supplement (and improve on) a popular laboratory handbook. With this example in mind a manual suited to the special needs of the National Collection of Type Cultures was prepared, and contributions were made by other members of the Collection staff, particularly Mrs P. H. Clarke, Miss H. E. Ross, Miss C. Shaw, and Mr C. S. Brindle. The National Collection Manual in turn became the basis for the appendices to the present Manual. In compiling the tables we sought information from various sources, including authoritative works such as the Reports of the Enterobacteriaceae Subcommittee of the International Committee on Bacteriological Nomenclature, and monographs such as Kauffmann's (1954) Enterobacteriaceae, Edwards & Ewing's Identification of Enterobacteriaceae (1962), and Smith, Gordon & Clark's (1952) Aerobic Sporeforming Bacteria. We found large gaps in published works, and in many instances

our own data have been the only source of information. While we have taken great care in compiling and checking the tables, we are sure that the Manual is unlikely to be free from error. When such errors are detected we hope that the finders will let us know. We will also welcome data to fill up the few gaps in the tables. It is with pleasure that we acknowledge our indebtedness to many friends and colleagues at home and abroad for facts and discussions that have helped to clarify ideas. It is impossible to name them all, but we could not have planned or written the Manual without the help of Dr R. E. Gordon, Dr P. R. Edwards, Dr W. H. Ewing, Dr T. Gibson, Dr Joan Taylor, Mrs P. H. Clarke, Miss C. Shaw, and Miss H. E. Ross. We also wish to thank Miss B. H. Whyte and Miss A. Bowman, the Colindale librarians, Miss M. I. Hammond who dealt skilfully with the manuscript, and Mr W. Clifford who made the figures. London S.T.C. 1964 K.J.S. POSTSCRIPT My colleague, Dr K. J. Steel, died suddenly on 25 September 1964, between the completion of the manuscript and the proof stage of the book. His death at the age of 34 is a great loss for he seemed destined to reach the highest branches of bacteriology. In this Manual he was responsible for the whole of Appendices A to D and F and for much of Chapter 3; and he played a big part in revising and recasting the tables that form the heart of our work. I hope that the book will serve as a fitting memorial to a great collaborator and friend. London S.T.C. 1965 XI

Preface to the first edition

Our 'Diagnostic Tables for the Common Medical Bacteria' were originally published in the Journal of Hygiene. The tables seemed to fill a need and the demand for reprints was so great that Cambridge University Press reprinted them in pamphlet form. Many inquired about the technical methods, and there were constant complaints that the methods were not described and that the text lacked details of the taxonomic problems. We resolved, therefore, to expand the original paper and to prepare a book which would give sufficient detail of media and methods to justify its description as a laboratory manual. Although designed for medical workers we hope that others will use it. The value of a laboratory manual was impressed on one of us in 1935 at the British Postgraduate Medical School. Dr A. A. Miles had prepared a loose-leaf mimeographed manual to supplement (and improve on) a popular laboratory handbook. With this example in mind a manual suited to the special needs of the National Collection of Type Cultures was prepared, and contributions were made by other members of the Collection staff, particularly Mrs P. H. Clarke, Miss H. E. Ross, Miss C. Shaw, and Mr C. S. Brindle. The National Collection Manual in turn became the basis for the appendices to the present Manual. In compiling the tables we sought information from various sources, including authoritative works such as the Reports of the Enterobacteriaceae Subcommittee of the International Committee on Bacteriological Nomenclature, and monographs such as Kauffmann's (1954) Enterobacteriaceae, Edwards & Ewing's Identification of Enterobacteriaceae (1962), and Smith, Gordon & Clark's (1952) Aerobic Sporeforming Bacteria. We found large gaps in published works, and in many instances

our own data have been the only source of information. While we have taken great care in compiling and checking the tables, we are sure that the Manual is unlikely to be free from error. When such errors are detected we hope that the finders will let us know. We will also welcome data to fill up the few gaps in the tables. It is with pleasure that we acknowledge our indebtedness to many friends and colleagues at home and abroad for facts and discussions that have helped to clarify ideas. It is impossible to name them all, but we could not have planned or written the Manual without the help of Dr R. E. Gordon, Dr P. R. Edwards, Dr W. H. Ewing, Dr T. Gibson, Dr Joan Taylor, Mrs P. H. Clarke, Miss C. Shaw, and Miss H. E. Ross. We also wish to thank Miss B. H. Whyte and Miss A. Bowman, the Colindale librarians, Miss M. I. Hammond who dealt skilfully with the manuscript, and Mr W. Clifford who made the figures. London S.T.C. 1964 K.J.S. POSTSCRIPT My colleague, Dr K. J. Steel, died suddenly on 25 September 1964, between the completion of the manuscript and the proof stage of the book. His death at the age of 34 is a great loss for he seemed destined to reach the highest branches of bacteriology. In this Manual he was responsible for the whole of Appendices A to D and F and for much of Chapter 3; and he played a big part in revising and recasting the tables that form the heart of our work. I hope that the book will serve as a fitting memorial to a great collaborator and friend. London S.T.C. 1965 XI

Preface to the second edition

The first edition of this Manual, judged by its spread around the world, seems to have been useful to hospital bacteriologists. It was translated into Japanese by Dr Riichi Sakazaki, who will also translate this edition. It has not been easy to prepare a worthy successor; not only have I been unable to discuss and argue every sentence with my colleague, but I have missed the ready access to libraries that one has when working in a large research institution. However, I have been greatly helped by the Librarians at Colindale (Miss B. H. Whyte) and the Royal Society of Medicine (Mr P. Wade) and their staffs. In this edition Chapter 2 and Appendices A, B, C and E, originally written mainly by Dr Steel, are little changed; most of the other chapters have been completely rewritten. Chapters 8 and 9 are entirely new, as are Appendices D, F, G and H. I must thank Mr A. Waltho, of the Medical Research Council's Central Store, who gave me great help in preparing the list of firms which supply media and chemicals (Appendix H) and, together with Dr O. M. Lidwell, suggested and drafted what became Table 2.1. I am also grateful to many other colleagues who gave me information and advice; while it is impossible to mention all by name, I am particularly indebted to G. I. Barrow, W. B. Cherry, E. A. Dawes, N. E. Gibbons, R. E. Gordon, R. M. Keddie, S. P. Lapage,

xn

H. Lautrop, J. Midgeley, M. J. Pickett, R. Sakazaki, R. Whittenbury and S. A. Wright. On behalf of the Executive Committee of the International Association of Microbiological Societies (IAMS), Dr N. E. Gibbons gave permission for the reproduction of the Introduction to the proposed revision of the Bacteriological Code (Appendix G), and I should like to express my thanks to the IAMS Executive. In a book with so many tables and cross-references it is inevitable that some errors and inconsistencies are still undetected; I hope that these will be drawn to my attention so that corrections can be made in later impressions. For the proof reading I am grateful for help from former colleagues, Miss H. E. Ross, Dr G. I. Barrow and Dr A. F. B. Standfast. Checking the numerous and large tables in the manuscript and proof stages has been an onerous task which I could not have done without the co-operation of my wife, who also helped to check the references, which must now number about a thousand. With all this help, I hope the book will continue to be a worthy memorial to my much missed young colleague, Dr K. J. Steel. Queen Camel 1973

S.T.C.

Preface to the third edition

The demand for a new edition of this Manual has been enormous. We hope that we have done justice to it and that it will prove a fitting tribute to the late Sam Cowan. He not only obtained every paper he cited in the references but personally perused and annotated each one. We cannot alas say the same. It is now beyond the scope of one person or even of two persons to cover the entire and seemingly everchanging fields of bacterial classification, nomenclature and taxonomy, especially with the range of 'medical' bacteria expanding with the advancement of biotechnology and modern medicine to include many environmental organisms. For this third edition we have therefore sought the help of the experts listed on pages xv and xvi for various groups of organisms and we gratefully acknowledge all their contributions to this Manual. The opinions expressed are mostly theirs though the final responsibility is ours. We hope that together they will provide enlightenment and understanding of a subject which, though not everyone's 'cup of tea', is nevertheless at the heart of diagnostic medical bacteriology. In outline, this edition follows that of the two previous ones. We have received numerous suggestions for change but have resisted many of them, preferring to regard continuity as more important. We have also retained references to some methods and equipment which may be regarded as 'old-fashioned' or not quite reaching the current acme of absolute safety, but we are conscious that not all diagnostic laboratories are equally endowed; we know for example that, despite their limitations, manually operated autoclaves are still used frequently and apparently satisfactorily throughout the world. Apart from extensive updating of the text, tables and appendices, we have added new Sections: on rapid methods and test kits; on the theory and use of computers for bacterial iden-

tification; on the principles of the Bacteriological Code and the Approved Lists of Bacterial Names; and on the reconciliation of different approaches to bacterial systematics. Unlike previous editions, we have not listed 'sources of information' separately in Chapters 6 and 7 but have included all references in the text. ,Also, in this edition we have omitted the Appendix listing some of the manufacturers of media, reagents and other laboratory supplies as the international names are now well known and we think it would be invidious to select arbitrarily from the many others. For reference purposes we have included the type strain of the type species in the minidefinitions, but we emphasize that they should not be regarded as necessarily 'typical' in all respects of the species. In a book such as this with so much material and so many cross-references we know that there are bound to be some errors and inconsistencies that we have missed; moreover with the increasing scope and application of genetic and other techniques, some of the taxonomic information will probably be out of date already. We should be glad therefore if readers would draw such occurrences to our attention for subsequent correction. Professor P. H. A. Sneath, who has himself contributed much to bacterial systematics and taxonomy, has kindly written a Foreword. On behalf of the International Union of Microbiological Societies Professor S. W. Glover gave permission to reproduce the Introduction from the International Code of Nomenclature of Bacteria and from the Approved Lists of Bacterial Names, and also the text of the Report of the Ad Hoc Committee on the Reconciliation of Approaches to Bacterial Systematics. We wish to express our thanks to him and to the IUMS executive. For proof-reading and checking so many tables, xni

PREFACE TO THE THIRD EDITION

we express our sincere thanks to Dr Joan M. Davies and, in particular to Dr B. Holmes who also contributed a large part of Chapter 7 on the Gram-negative bacteria and made many useful suggestions as did Dr Dorothy Jones. We also thank our many other colleagues, too numerous to name, for their help in many if unspecified ways. As before, Dr R. Sakazaki will translate this edition into Japanese and we are grateful to him for this. Last but not least we thank

xiv

our wives and the staff of Cambridge University Press for their forbearance and support in what proved to be a long and arduous task. We hope that this new edition will be at least as useful as the previous two seem to have been, Salisbury Leicester 1992

G.I.B. R.K.A.F.

Contributors

G. Colman Division of Hospital Infection, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT. E. Foxt Public Health Laboratory, Leicester Royal Infirmary, Leicester LEI 5WW. R. J. Gross Division of Enteric Pathogens, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT. B. Holmes Identification Services Laboratory, National Collection of Type Cultures, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT. P. A. Jenkins PHLS Mycobacterium Reference Unit, University Hospital of Wales, Heath Park, Cardiff CF4 4XW. Dorothy M. Jones Department of Microbiology, Medical Sciences Building, University of Leicester, University Road, Leicester LEI 9HN. J. V. Lee PHLS Environmental Microbiology Reference Unit, Public Health Laboratory, Queen's Medical Centre, Nottingham NG7 2UH. R. R. Marples Division of Hospital Infection, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT. F. G. Priest Department of Biological Sciences, Heriot-Watt University, Riccarton, Edinburgh EH14 4AS. ^ deceased XV

LIST OF CONTRIBUTORS

Geraldine M. Schofield Unilever Research Laboratory, Colworth House, Sharnbrook, Bedford MK44 1LQ. M. W. Scruton Sterilization R&D Unit, PHLS Centre for Applied Microbiology & Research, Porton Down, Salisbury, Wiltshire SP4 OJG. M. B. Skirrow Public Health Laboratory, Gloucester Royal Hospital, Gloucester GLl 3NN. Mary P. E. Slack Department of Bacteriology, John Radcliffe Hospital, Headington, Oxford 0X3 9DU. J. J. S. Snell Quality Assurance Laboratory, Division of Microbiological Reagents and Quality Control, Central Public Health Laboratory, 61 Colindale Avenue, London NW9 5HT. A. T. Willis Public Health Laboratory, Luton & Dunstable Hospital, Lewsey Road, Luton LU4 ODZ.

xvi

Introduction

It is assumed that the reader of this Manual has some knowledge and experience of bacteriology and of elementary chemistry and that the basic principles including those of laboratory safety are understood. Thus, though many other essential details are given in the Appendices, how to determine the pH value of a medium, or how to make a normal or molar solution, is not described; nor are details given about how to use anaerobic jars or microscopes. Serology is not discussed but methods commonly used in the preparation of extracts for grouping streptococci are described as the Lancefield serological groups are referred to in Table 6.3b. Details of sterilization temperatures and times are also given as these so-called standard procedures still vary from one laboratory to another. This Manual is intended to help those who have isolated a bacterium and want to identify it. The methods used by clinical bacteriologists to isolate organisms from specimens sent to the laboratory are not described as to do so would be to enter everchanging fields, and our recommendations might well be out of date. We stress, however, that before identification of any organism is attempted, it must be obtained in pure culture. Some advice on how to recognize that a culture is impure, and on the steps to be taken to purify it, is therefore given. The tables for identification of medical bacteria developed in phases: the original tables of Cowan & Steel (1961) were based mainly on the results of tests carried out on strains in the National Collection of Type Cultures (NCTC) between 1948 and 1960. For the tables in the first edition of this Manual, the NCTC information was supplemented by surveys of the literature up to 1963; the second edition included the results of further literature surveys up to 1972; and for the present (third) edition, the literature up to

1990 has been reviewed by individual experts for each of the principal bacterial groups; where possible, new genera subsequently accepted, such as Enterococcus and Helicobacter, have been included. Each expert was asked to provide identification tables and methods suitable for use in routine diagnostic laboratories. It follows that in this edition we are less often able to indicate the relative value of the different technical methods used to obtain the characters shown in the tables. Once again, discrepancies occurred between the results of different workers probably due more often to differences in methods than to variation between strains of the same species. To try to cover every possibility in the tables would be self-defeating, for either we multiply the columns (species or varieties) or we increase the number of equivocal or doubtful entries (d or D) equivalent to words like 'often', 'some(times)', or 'not infrequently', so that a clear positive or negative character would become rare and the tables thus confusing and unhelpful. We were tempted to use the percentage of positive results, as utilized for computer-assisted identification, but after careful consideration we felt that this would complicate the tables unnecessarily and be helpful only to a minority of readers. We have tried therefore to be definite and have treated descriptions such as 'occasionally', 'occasional strains' and 'a few strains' as exceptions not worthy of note. The tables are therefore not perfect as there are exceptions to all rules: it is the user who must be realistic and bear in mind that the bacteria they are trying to identify may not conform to the expected norm. Intelligent use of the tables demands technical skill and sensitive but specific methods for the individual tests. As in all determinative bacteriology, true identification must be based on careful work, and the tables will not help those who are in too much of a hurry to xvn

INTRODUCTION

carry out the basic tests needed, though this does not necessarily mean every test in a table. We considered cutting down the tables to show only those characters that had immediate value in distinguishing one species from others in the tables, but we decided against this because conditions vary considerably in different laboratories and in different countries. We do not expect all the tests in the tables to be carried out; each bacteriologist has individual preferences and dislikes; and not all laboratories are equally well equipped. For these reasons many more characters than are necessary for identification have been included. We considered indicating the more important characters in bold type, but as this would merely reflect our own preferences we decided against it. Bacteriologists must choose those tests that seem to them to be most discriminating and use those that can be performed with the equipment and media available. As the tables are constructed from information from many sources, particular methods are not stipulated, but those given in the Appendices should be satisfactory. In this edition we have omitted many of the micromethods described in the first edition; those retained are included in Appendix C, together with methods using larger volumes (and often taking a longer time). Three points should be emphasized about the tables, (i) They should not be considered in isolation; other evidence that cannot be included in them such as colony form, experimental pathogenicity, chromatographic profiles, chemotaxonomy and DNA hybridization results should also be taken into account, (ii) The tables do not characterize an organism; they are intended to focus attention on tests and characters most valuable in differentiation, (iii) The tables do not form part of any classification system, but they may draw attention to bacterial similarities and relationships that are not otherwise apparent. We have not been able to avoid taxonomic terms completely but a brief glossary of those in current use is included at the end of this Manual; for further information, Cowan's Dictionary of microbial taxonomy (Hill, 1978) should be consulted. Names of species are not shown in the table headings but as numbered footnotes, and these include common synonyms so that, with the Index, it should be possible to find the main characters of many named species. The definitive names given in the xvin

new Approved Lists of Bacterial Names (Skerman, McGowan & Sneath, 1980) including validated changes and additions subsequently published in the International Journal of Systematic Bacteriology (IJSB), are used throughout. The older generic name 'Bacterium' is not included and the term Bacillus is restricted to aerobic spore-formers. In general, the tables allow identification of species that can often be further differentiated into serotypes, biotypes or phage types. However, users of the Manual will seldom have all the sera needed for detailed antigenic analysis of the species they isolate; this is a task for a reference laboratory. Those who aspire to do such work themselves should consult the excellent practical manual by Edwards & Ewing (1962, 1972) which highlights the problems and, for the Enterobacteriaceae, gives the essential details. The tables seldom mention sensitivity or resistance to antibiotics although sometimes such tests are of practical differential value. In the present era, however, apart from selection pressure, the genetic effects of antibiotic therapy and usage are known to affect bacterial characters, including sensitivity or resistance to antibiotics, by transfer of plasmids. We have tried to refer readers to pertinent literature in which fuller details of methods are given; in this way we have kept the Manual free from unnecessary detail and from the more theoretical aspects of taxonomy and nomenclature. We have, however, tried to retain where possible some of the nomenclatural life stories of many organisms, lest they be forgotten. In plan, the Manual falls into two parts: the first, divided into chapters, is discursive; the second, made up of appendices, is instructive and written tersely with free use of scientific abbreviations (Ellis, 1971), chemical formulae, and prescription-like recipes for media. The essence of the book is in Chapters 6 and 7, which comprise the diagnostic tables and notes on the different genera. In this edition we include more little-known genera, many of them incompletely defined. It may seem that we have sometimes strayed outside the medical field, but many environmental organisms are now becoming important as opportunist pathogens especially with the widespread use of immunosuppressive therapy and the advent of AIDS and the Human Immunodeficiency Virus. A practical example is given of the application of the diagnostic tables to punched cards for easy

INTRODUCTION

sorting and bacterial identification. Reference is also made to the use of computers for rapid comparison and identification of isolates. In addition, the Manual now includes a short chapter on the quality control of

laboratory procedures and reagents as well as on quality assessment, both within and between laboratories, with simulated material of known but undisclosed content.

xix

Classification and nomenclature

Taxonomy is not every man's meat, but neither is it everyone's poison. It can be likened to a cocktail: a skilful blend in which it is not easy to discern the individual ingredients. In taxonomy the ingredients are (i) classification, or the orderly arrangement of units, (ii) nomenclature, the naming or labelling of the units, and (iii) identification of the unknown with a unit defined and named by (i) and (ii). The subdivisions should be taken in the order indicated, for without adequate classification it is impossible to name rationally, and wijhout a system of labelled units it is impossible to identify others with them or to communicate the results. 1.1

Classification

Before discussing the identification of bacteria, the principles of classification and nomenclature must first be dealt with briefly. Since this book is essentially a practical manual, theoretical speculations about the validity of bacterial species (Lwoff, 1958; Cowan, 1962a; Lapage et a/., 1975) are not considered. For this Manual, the concept of bacterial species is therefore accepted as a convenient unit. However, as it so obviously has different values in different groups of bacteria, no attempt is made to define it, or to analyse the qualities that distinguish one species from another. Nor is any attempt made to determine whether a taxonomic group (taxon) is a species, a variety or a subspecies; or to estimate the value or importance of different kinds of bacterial characters (Cowan, 1968, 19706). We dislike the idea of subspecies and prefer to recognize subdivisions of species either as varieties (biotypes) or as serotypes. We do not accept the contention of Kauffmann (1959a, b\ 1963&) that the serotypes or phage types of the various members of the Enterobacteriaceae

should be equated with species. However, the collection of similar species into larger groups (genera), and similar though not necessarily related genera into families, are convenient and generally accepted groupings. But they should not be regarded as phylogenetic groups; thus to combine families into even larger groups (orders) would be artificial and highly speculative. The different kinds of bacteria are not separated by sharp divisions but by slight and subtle differences in characters so that they seem to blend into each other and resemble a spectrum (Fig. 1.1a, b). This spectrum-like intergrading of different kinds of bacteria is confirmed by other methods of grouping, such as the base composition of the deoxyribonucleic acid (DNA) of the bacterial cell (Vendrely, 1958). The DNA composition varies among different groups of bacteria but it should be homogeneous within a group. Marmur, Falkow & Mandel (1963), Hill (1966) and Brenner (1978) collected the results of numerous workers and summarized in tables the DNA-base composition and DNA homology of many bacteria. These techniques are not applicable in dayto-day diagnostic work but the results are of fundamental importance to the taxonomist; the essential aspects are considered in a recent Report (Wayne et aL, 1987) on the reconciliation of different approaches to bacterial systematics. The recent introduction of cloned DNA as probes in diagnostic bacteriology is not only very exciting but may well revolutionize diagnostic bacteriology as currently practised. Indeed, diagnostic DNA probes have already been developed for many of the pathogenic bacteria as well as for several viruses. A theoretical classification scheme divides the higher ranks into two or more kinds of a lower rank; for example, earlier editions of Bergey's Manual of Determinative Bacteriology (1923-57) divided the 1

[1.1]

CLASSIFICATION AND NOMENCLATURE

(b) Gram-negative genera

(a) Gram-positive genera "Staphylococcus

Bacteroides

—

Edwardsiella

"Micrococcus

Fusobacterium

—

Enterobacter

—

Mobiluncus

—

Erwinia

Acidaminococcus

—

Escherichia

Leptotrichia

"Stomatococcus

Veillonella

"Aerococcus 'Streptococcus

Megasphaera

—

Hafnia

- Enterococcus

Branhamella

—

Klebsiella

-Lactococcus

Neisseria

—

Kluyvera

-Leuconostoc

Acinetobacter

—

Morganella

—

Proteus

—

Providencia

—

Salmonella

—

Serratia

—

Shigella

—

Tatumella

—

Yersinia

—

Budvicia

—

Ewingella

—

Koserella

Kingella

-Gemella

7.4

Brucella

-Peptococcus

Bordetella

-Peptostreptococcus

Alcaligenes

-Corynebacterium

Shewanella

-Listeria

Pseudomonas

7.9 7.5

Achromobacter

-Brochothrix

Ochrobactrum

-Kurthia

Agrobacterium

-Lactobacillus

Janthinobacterium

7.6

-Erysipelothrix

Flavobacterium

—

Leminorella

-Arachnia

Chryseomonas

—

Moellerella

-Actinomyces

Flavimonas

—

Obesumbacterium

Weeksella

—

Pragia

Pasteurella

—

Rahnella

—

Xenorhabdus

-Arcanobacterium -Propionibacterium

Actinobacillus

7.7

-Rothia

Cardiobacterium

—

Haemophilus

-Bifidobacterium

Francisella

—

Gardnerella

'Clostridium

Vibrio

—

Eikenella

-Eubacterium

Aeromonas

—

Campylobacter

—

Helicobacter

—

Arcobacter

—

Anaerobiospirillum

—

Streptobacillus

-Bacillus -Mycobacterium

6.10

7.3

Moraxella

-Pediococcus

6.9

12

Plesiomonas

7.8

Chromobacterium Buttiauxella

-Nocardia

Cedecea

-Actinomadura

Citrobacter

7.9

7.10

Legionella

Fig. 1.1 (a) Gram-positive and (b) Gram-negative genera dealt with in this manual.

CLASSIFICATION

kingdom Bacteria into orders and continued the breakdown through families, tribes, genera, and species; however, neither the last (8th) edition (1974) nor the current volumes of Bergey's Manual of Systematic Bacteriology (1984, 1986, 1989) attempt to produce a complete hierarchy. We suggest, as in previous editions of this Manual, that a pragmatic classification should be built up from the basal unit (species); basal units which share a number of characters are combined to form the next higher unit (genus) so that the common characters become those which are important in the definition or characterization of the genus. The emphasis on certain characters - which are regarded as important because they are significant in identification - differs from the Adansonian concept now referred to as numerical taxonomy (Sneath, 1957a), in which each character has equal merit in the eyes of the taxonomist. What are called 'important characters' may be of three kinds: (i) specific, such as the ability to produce coagulase by Staphylococcus aureus; (ii) distinguishing characters which, though not specific, are useful in separating organisms that are otherwise very similar (for example, indole production by Proteus vulgaris is one of the characters that distinguishes it from P. mirabilis); (iii) characters shared by all members of a group; thus, acid-fastness is an important character of mycobacteria since all members are acid-fast, but not of the nocardias only some of which are acid-fast. Needless to say, there are degrees of importance and it would be feasible to continue the list; but enough has been said to illustrate the point that characters can have an importance in a pragmatic scheme that are denied to them in numerical (Adansonian) classification. Taxonomists use and put much weight on what are called 'fermentation tests' without paying too much attention to the way in which carbohydrate is broken down; this breakdown is characteristic of an organism and of great informative value for classification. In this Manual the terms 'oxidative' and 'fermentative' are used though it is not at all certain that they describe accurately what is happening in the Oxidation-Fermentation (OF) test of Hugh & Leifson (1953). A practical classification should preferably be based on characters that are easily determined; consequently features that demand difficult techniques or

[1.1] special apparatus should not be used. Thus, bacterial components such as cell walls, septa, nuclei, and fimbriae are excluded from our scheme despite their importance, though to add them would increase the weight of our argument and support our general conclusions. Again, because it is difficult, even with an electron microscope, to obtain accurate pictures of the arrangement of flagella on bacteria, little weight is placed on this character though it is very important in all except the most rigid numerical taxonomy. The scheme used in this Manual is built on a wide range of characters; the identification of each unit is based on the same characters and it is not necessary to make the hypothetical and sometimes absurd assumptions of classical taxonomy. By omitting any reference to the type of flagellation, there is no need to postulate that Shigella species, if they had any, would have peritrichous flagella. We do not use any formal classification scheme in this Manual but, because of the need to label recognizable taxonomic units, usually at the species level, advantage is taken of the specific epithets and generic names that are already part of formal systems. Labelling is discussed more fully in Chapter 8; only the problems that immediately concern the identification of bacteria, and how the organisms are to be described in reports to clinicians and health officials, are mentioned here. It is convenient to divide bacteria into two large groups based on the reactions of the organisms to Gram's method of staining, though latinized names are not given to these groups. In formal schemes most genera consist of bacteria that are either Gram-positive or Gram-negative, so that each genus can be allocated according to the Gram reaction of the majority of species it contains. The genera can be arranged in some order so that adjacent ones show some similarities but the more distant genera have less in common (Fig. 1.1a, b)\ the arrangement resembles two series of pigeon-holes - one for Gram-positive and the other for Gram-negative organisms - where the partitions between the individual pigeon-holes are removable so that the contents of adjacent holes will be in contact with each other. But although bacteria may be regarded as a series of gradually merging forms and likened to a spectrum, we do not imply that the

[1.1]

CLASSIFICATION AND NOMENCLATURE

sequences shown in Fig. 1.1a, b are the best that can be devised to show relationships, if present, between the genera. The sequences have some merit in that they do not violate too severely the order that might be presented in a classification made on orthodox lines. We stress therefore that the lists of names in Fig. 1.1a, b are those of taxa that are normally regarded as having generic rank; they are not an attempt at classification. Most of the names have been in use for a long time and are generally accepted. With some bacteria the Gram reaction is regarded as variable, indeterminate, and occasionally misleading. Many cocci and some rods may be Gram-positive in young cultures and become negative as the culture ages; in such cases it is usual to regard the reaction of the young culture as correct. Occasionally the reverse sequence occurs: Acinetobacter strains for example may become somewhat Gram-positive in cultures several days old and some workers (e.g. Thornley, 1967) describe them as Gram-variable; this is a phenomenon better known outside the medical field, as in the genus Arthrobacter. Strains of Gemella are unusual in that when stained they seem to be Gram-negative, but in the chemical nature of the cell wall they resemble the Gram-positive bacteria. Although cell wall analysis is far from a routine procedure, we have placed the genus Gemella in Fig. 1.1a among the Gram-positive bacteria but show the characters of the species in tables dealing with both Gram-positive (Table 6.3a, b) and Gram-negative (Table 7.3) cocci. Because we do not start with an orthodox classification in a hierarchical system, it may be argued that the identifying characteristics of bacteria cannot be presented in a logical manner. However, by avoiding a formal classification we believe that the characters can be displayed in a manner that is both logical and orderly; that similarities and differences shown by the tables will point the way to a more orderly arrangement of the different taxa; and, as orderly arrangements are the essence of all classifications, the tables may thus lead to a logical classification. It is essential that the reader should understand that there is no single classification ordained by God or by Nature but numerous different classifications, all made by man, each with a particular purpose in mind. The main difficulty without a classification relates to the labelling of the taxa; this difficulty is

overcome in our tables by using the names(s) by which each taxon (usually a species) is generally known, irrespective of the lightness of the name(s) in terms of the International Code of Nomenclature of Bacteria. The taxa listed in Fig. 1.1a, b are groups approximating to genera and the components of these groups will be species whose characters are shown in the secondary and tertiary tables in Chapters 6 and 7. Usually the acceptable name for a group will be obvious and well known. The Gram-positive and Gram-negative bacteria can each be considered as a continuous series. Some of the genera shown in Fig. 1.1a, b are dealt with only summarily in this Manual but they are mentioned because they may be met either as contaminants or as suspected pathogens. Organisms such as Leptospira and other spirochaetes that are identified on morphological and serological grounds, or by their pathogenicity for various animals, do not fit into the scheme in this Manual. Neither do mycoplasmas, though they are referred to briefly in Section 7.10.10. Our purpose is to identify bacteria isolated in medical laboratories, commonly, if incorrectly, called the medical bacteria. They are derived mainly from clinical material (hospital laboratories) but may come from apparently healthy individuals (public health laboratories), or from water, sewage, foodstuffs and other environmental sources. Apparently identical bacteria may produce infection in animals other than man, so our survey must include the fields of medical and veterinary science, and it will necessarily impinge on plant pathology and industrial processes. 1.2

Nomenclature

In this Manual, emphasis is placed on the characterization of organisms, for it is a waste of time to work out carefully the characters of the unknown and then compare these with vague descriptions. We do not make a great point about nomenclature, believing that a simple label, as long as it is unique, is adequate for communication though we abide by the Approved Lists of Bacterial Names (Skerman, McGowan & Sneath, 1980). In the tables in Chapters 6 and 7, the columns of characters of a taxon are numbered and the name(s) by which the taxon is known will be found in numbered footnotes. The first name, usually in bold type, is generally regarded as the most accept-

NOMENCLATURE

able name; this is usually, but not necessarily, the nomenclaturally correct name. It is important that everyone concerned - the bacteriologist, the clinician, and the health official - should all understand fully the nature of the organism reported. The nomenclaturally correct name often means little outside the laboratory and those in charge of patients may still be blissfully unaware of the implications; on the other hand, the common name in English may be understood more readily. In the tables we give, for good measure, other names used in English- and American-speaking countries; these synonyms appear in the footnotes without further explanation. Latin and latinized names in the text are avoided as far as possible. In this chapter, the problems and principles of nomenclature are not discussed, but we must point out now that, as nomenclature is dependent on classification, there may be more than one correct name for a bacterium. Nomenclature is subject to the rules of the International Code of Nomenclature of Bacteria, usually referred to as the Bacteriological Code (1976 revision); there are not, and never can be, any rules for classification. Classification is subjective and a matter of opinion, and it is within the rights (if not the competence) of each worker to classify bacteria as he or she will. The classification adopted determines the names to be appended to the organisms, and the rules of nomenclature should be a guide to the choice of correct names. If a worker believes that all rod-shaped organisms are to be grouped together in one genus, Bacillus, he is entitled to name the diphtheria organism 'Bacillus diphtheriae', but if he thinks that rod-shaped bacteria can be split into different groups he can use a name such as Corynebacterium diphtheriae; each name is correct within its own taxonomic scheme or classification, and would be wrong in the other scheme. Regrettably, the application of the rules of the Bacteriological Code is still subjective and different workers may draw quite different conclusions from reading the rules and may interpret them in contrasting and even conflicting ways. One of the aims of the Bacteriological Code was to stabilize nomenclature; but this is an impossibility for nomenclature is itself dependent on ever-changing ideas on classification. The rediscovery and application of ah old or the development of a new

[1.2] technique may indeed act as a stimulus. Sometimes, an organism remains unclassified for decades after its discovery and characterization. Morgan's no. 1 bacillus (Morgan, 1906) needed the insight of Rauss (1936) and the discovery that under the right conditions it could be made to swarm to establish it as a species of Proteus. It was the rediscovery and application of the phenylalanine test that made Singer & Bar-Chay (1954) realize that Stuart's 29911 (Stuart, Wheeler & McGann, 1946) or Providence group was also a species of Proteus. With the advent of DNA hybridization techniques, both these organisms have now been reclassified and placed in the genera Morganella and Providencia respectively. It is often easier to create a new genus or species than to do the comparative work necessary to put an organism into its rightful place in an existing genus or species. The temptation to designate a new genus or species should be resisted, as it would be if it was appreciated that taxonomic ability is judged as inversely proportional to the number of new taxa created. Many workers use common names (in the vernacular language of their own country) in preference to scientific names that may be subject to change. Since English is now the language of science, common English names are widely understood, but French or German equivalents may present difficulty to English-speaking people (e.g. bacteridie de charbon = der Milzbrandbazillus = the anthrax bacillus = Bacillus anthracis). The great advantage of the latinized binomial name is that it is accepted throughout the world and the same words should have the same meaning everywhere. Although printed in the same way, even in journals using pictorial characters, the sound and pronunciation can, however, differ considerably. Nomenclature often presents difficulties because a change in name may be necessary when an organism is moved from one group to another. Sometimes the nomenclatural difficulties arose because the rules in the Bacteriological Code, first published in 1948 (Buchanan, St John-Brooks & Breed, 1948), revised and annotated in 1958 (Buchanan et al., 1958) and revised again in 1966, were made retroactive. This meant applying the rules to names first used in the last century, long before a bacteriological code was

CLASSIFICATION AND NOMENCLATURE

thought of, and in some instances, before the organism named had been isolated and characterized. Fortunately, following initial proposals by Lapage et aL, (1973) the rules in the Bacteriological Code were revised and simplified in 1975 (1976 revision) by the International Committee on Systematic Bacteriology which also reviewed bacterial nomenclature critically and published Approved Lists of Bacterial Names in 1980. This will do much to avoid petty squabbles on the priority of names in future. Another source of confusion is due to the wellintentioned efforts of workers to give a meaning to the specific epithet, and to make the epithet appropriate. For example, an ill-conceived attempt was made about 50 years ago to apply the epithet 'pyogenes9 to the generic name Staphylococcus because the legitimate epithet 'aureus9 was inappropriate for strains that produced white colonies. More recently Foster & Bragg (1962) suggested that various specific epithets for Klebsiella, correctly proposed by the old Rules, should be transposed (which would greatly add to confusion) because, as originally proposed, they seemed to them to be inappropriate. Yet another source of confusion is the re-use of a discarded name for a newly described genus or species. An example was the use of 'Aerobacter9 (a

[1.2] later synonym for Klebsiella) for a group of motile organisms which share several characters in common with non-motile organisms then named 'Aerobacter aero genes9. This confusion was later remedied by its authors (Hormaeche & Edwards, 1960) who withdrew their proposal and substituted the new generic name Enterobacter. Unfortunately so many authors continued to use the name Aerobacter for both motile and non-motile organisms that the problem was submitted (Carpenter et al, 1970) to the Judicial Commission of the International Committee on Systematic Bacteriology for an Official Opinion on the validity of the name Aerobacter; the Commission ruled (Opinion 46, 1971) against the use of the generic name Aerobacter because its application was uncertain. This kind of situation should not occur with the Approved Lists of Bacterial Names (Skerman, McGowan & Sneath, 1980) provided the rules are followed carefully and thoughtfully. We do, however, recommend caution in applying the results of new techniques such as DNA hybridization to the reclassification of existing species. A new name, although perhaps valid for DNA relatedness, is of little use if the same organism cannot be readily differentiated from close neighbours by routine clinical laboratory methods.

Culture media: constituents and sterilization

In the introduction to a Good Food Guide for bacteria, Miles (1965) described most culture media formulations as 'kitchen recipes, written ... by increasingly sophisticated cooks'. There are two entirely different kinds of media: those of defined composition and those of undefined composition, usually containing peptone. Defined media have disadvantages for identification because the characters of organisms grown in them may differ from those developed in undefined media (Meynell & Meynell, 1965). In general, published descriptions of bacteria refer to those characters found after growth in complex, undefined media, and because of this the results of biochemical tests do not always correspond with those obtained in defined media. Media preparation seldom receives the attention it deserves; moreover, the media room is often overcrowded and understaffed, and the conditions in which media-makers work are often among the worst in the laboratory. Complaints about media, whether commercial or home-prepared, are still common and many laboratories have therefore set up internal quality control and assessment procedures (see Chapter 11); others have also made formal arrangements for supervision of media preparation. In this chapter we discuss the general aspects of media making; formulae for the different media will be found in Appendix A. The majority of the commonly used culture media are now available commercially as dehydrated products, in either powder or tablet form, which are reconstituted by the addition of distilled water and then sterilized in the conventional manner. The manufacturers' directions for reconstitution should aways be followed for the best results. The advantages of dehydrated media include: ease of preparation usually without the necessity for pH adjustment or phosphate removal; batch to batch uni-

formity, which is often greater than with laboratoryprepared media; convenience for the preparation of small quantities; ease of storage; and economy especially in saving time and labour. Against these must be set certain disadvantages. Most of the dehydrated products are hygroscopic but this is usually overcome by the use of plastic or foil sachets containing sufficient for one batch of medium. Dehydration is not suitable for media containing blood, other thermolabile components, or egg. Another consideration where dehydrated media are widely used is that technical staff may not receive adequate training in the principles of media preparation. Although ordinary media may be used for the growth of obligate anaerobes, better growth usually occurs if the redox potential (£ h ) of the medium is reduced before inoculation. Ideally, reduction should be made during preparation of the media, the reduced conditions being maintained during storage and, as far as possible, during inoculation. The specialized techniques used for such 'pre-reduced anaerobically sterilized (PRAS) media' are described fully by Holdeman, Cato & Moore (1977). There are three essential steps in the preparation of PRAS media: (i) driving off dissolved oxygen by boiling, (ii) addition of cysteine, and (iii) flushing with oxygen-free gas and storing in stoppered tubes or bottles containing oxygen-free gas. These media contain an £/ h ) indicator (resazurin) which is colourless when reduced; if oxidation occurs, it becomes pink, indicating that the medium should not be used. Cysteine inhibits some proteolytic enzymes, and so should not be added to media for tests of this property. There is little doubt that roll-tube techniques such as those described by Hungate (1969) and Moore (1966) provide one of the best methods for the isolation and study of organisms which are highly sensi-

CULTURE MEDIA: CONSTITUENTS AND STERILIZATION

tive to oxygen. PRAS media prepared in roll-tubes are inoculated in the complete absence of oxygen, and all subsequent manipulations are performed in a stream of sterile oxygen-free gas. The value of this method in some non-clinical areas of anaerobic bacteriology is beyond dispute but it is not universally used in clinical work in the UK, mainly because of the special apparatus and techniques required. 2.1

Media for different purposes

Not all media are intended to encourage the growth of all bacteria; some media are tailored to suit the organism(s) they are able to encourage, and others are deliberately made unsuitable in order to inhibit the growth of certain (specified) organisms. 2.1.1 Isolation media. These may be simple nutrient media containing all essential constituents for growth; in medical laboratories the commonest general purpose medium is Blood Agar, but Chocolate Agar may often be more successful in the isolation of fastidious bacteria. The nature of the specimens received for examination is usually known and, with intelligent anticipation of the probable bacterial flora, media are chosen to suit the nutritional requirements of the organisms which can reasonably be expected to be present. For some bacteria the medium is made from constituents of known composition (that is, a defined medium) but most organisms isolated from clinical material are exacting and need the basal nutrient medium to be enriched or supplemented by substances such as serum, blood, haemin and vitamin K. Thus, media used in medical and veterinary laboratories are rich in unspecified proteins, and they encourage the growth of both wanted and unwanted organisms. 2.1.2 Selective or inhibitory media. When the specimen is from a part of the body (skin, throat, mouth, nose, intestine, vagina) with a natural microbial flora, growth of the normal inhabitants may be inconveniently profuse and the bacteriologist will want to limit or suppress such commensals but at the same time encourage growth of the invaders which, in clinical pathology, are the 'wanted' organisms. For this, selective or inhibitory media are used and as the inhibitory properties may be specific, the media must 8

[2.1]

be chosen with some particular organism (or group of organisms) in mind. There is no medium exclusively or universally selective for all pathogens, and it might seem that success in isolation depends on accurate prediction of which bacteria are to be inhibited and what requirements are needed for those that are to be encouraged. Since prediction is usually difficult, most workers increase the chances of isolating the organisms wanted by using several kinds of selective media* 2.1.3 Enrichment media. Usually both selective and inhibitory, these are liquid media into which swabs or specimens are placed; after incubation for 6 and 18 hours, subcultures are made to plates of (i) selective, and (ii) non-inhibitory nutrient media (Nutrient Agar, Blood Agar). After incubation these plates are examined and selected colonies subcultured to non-inhibitory media. This second plating is an important step in the isolation process; without it the colonies first subcultured may well yield a mixture of wanted and unwanted organisms. Whenever possible, selective media should be avoided; repeated plating on non-inhibitory media is preferable, although this is a council of perfection seldom satisfied in practice. 2.1.4 Media for the maintenance of cultures. These are simple and should not encourage luxurious growth; Nutrient Agar (in its many formulations) is the commonest (see Lapage, Shelton & Mitchell, 1970). For the preservation of serological characters, Dorset Egg, in spite of its imperfections, seems to be the medium of choice. Robertson's Cooked Meat medium is a good all-round maintenance medium, especially for clostridia. Simple media, such as Nutrient Broth, containing glycerol, can be used for freezing bacteria at temperatures as low as -76 °C (Feltham et aL, 1978). 2.1.5 Media for determining nutritional requirements or ability to use a single substrate are now being used increasingly in diagnostic work. To avoid chemical contaminants, agar of the highest purity should be used for solid media. When the nutritional requirements of an organism are unknown, omission of one component at a time and substitution of another may be used to identify which substrates

[2.1]

MEDIA FOR DIFFERENT PURPOSES

are essential for growth and which can be replaced by others. This is a time-consuming process not used in routine work. But in a simplified form, as in Koser's Citrate medium, a test for the ability to use citrate as a source of carbon is a useful characterizing test for enteric bacteria. For non-exacting bacteria, a basal medium inadequate for growth of the organism is used and one substrate at a time added tp find out whether the organism can use the substrate as a source of carbon or nitrogen. A suitable mineral base of this kind was described by Owens & Keddie (1969) and Cure & Keddie (1973). For the value of these tests, see Snell &Lapage(1973). Chemically clean glassware is essential for tests used to determine the nutritional requirements of an organism. Dirty glassware can provide nutrients; it may also be contaminated by substances inhibitory to growth (see Appendix Al.l). 2.1.6 Media for bacterial characterization generally consist of a simple but nutritionally adequate base to which thesubstrate under test has been added. Sometimes an indicator is included to show that a change in reaction has occurred; in other cases reagents are added after a specified period of incubation. 2.1.7 Screening media. These are intended to show at a glance the reactions obtained with several substrates, hence the term multitest media; they can be relied upon only when pure cultures are used. For this reason, the Knox (1949) plate, described as a screening plate, cannot be used for the original specimen but only for subcultures. Some multitest media are better used as a standard, relatively insensitive method for one of the reactions, as with TSI for H2S production by enterobacteria. 2.1.8 Media for microbiological assay of vitamins and amino acids need stringent control during preparation to ensure freedom from impurities; such media are available from commercial sources. The assay of antibiotics and the sensitivity testing of microorganisms do not need media of such rigid specification and can usually be carried out on ordinary nutrient media, with or without indicator.

2.1.9 Non-nutrient basal media. Occasionally a clear solidifying medium without nutrient properties is useful. Such a medium is needed for the top layer of Chitin Agar, the insoluble chitin being suspended in Water or Salt Agar. Saline Agar has other uses. Physiological saline solidified with agar may be used to line tubes for the collection of rabbit blood. After the blood has clotted, the agar lining retracts with the clot, and the serum, expressed through the agar, is water-clear. Such serum is anticomplementary. Non-nutrient media are widely used for transport purposes. 2.2

Media constituents

A fuller account of the derivation and properties of peptone, meat extract, yeast extract, gelatin, agar and bile salts is given in the Special Report of the Society for General Microbiology on constituents of bacteriological culture media (Report, 1956a); the design and formulation of media were reviewed by Bridson &Brecker(1970). 2.2.1 Agar can be obtained as shreds, flakes, granules or powder and is made from certain types of seaweed. The usefulness of its unusual gelling properties for bacteriological work was recognized by Frau Hesse, who suggested its use to her husband, Walther Hesse, an early colleague of Robert Koch (Bulloch, 1938; Hitchens & Leikind, 1939). When mixed with cold water, agar does not go into solution; it can therefore be washed to free it from soluble impurities. The concentration for use depends on the geographic source of species of seaweed from which the agar is made, and on the purpose for which the medium is intended (Appendix A, Table A5). In this Manual, the concentration of agar given in the formulae for media relates to the product derived from Japanese seaweed. In addition to the agar concentration, other factors affect gel strength; for example, repeated melting of the medium or prolonged sterilization especially at a low pH value will decrease it. 2.2.2 Peptone is a product of varying composition made by acid or enzymic hydrolysis of animal or vegetable protein, from material such as muscle,

CULTURE MEDIA: CONSTITUENTS AND STERILIZATION

liver, blood, milk, casein, lactalbumin, gelatin and soya bean. The exact composition depends on the raw material and the method of manufacture. No two batches of peptone are exactly alike, but commercial firms try to produce peptones in which the measurable constituents are present within certain defined limits. For many kinds of media the make or type of peptone is immaterial, but for certain tests a particular type may be specified. This does not mean that all other types are unsuitable; more often than not it means that other peptones may not have been tried. Certain batches of peptone, however, may be quite unsuitable for a particular purpose, and before general use a peptone should be tested. In the section on media control (Appendix A3) we discuss this problem in more detail and give examples of fallacious results due to the use of unsuitable peptones. Most peptones from reputable commercial sources are equally good. 2.2.3 Meat. Beef heart, muscle, and liver are commonly used but calf brain, veal, spleen, and placenta also have a place in media preparation. The quality of meat and other tissues varies with the age and health of the animal and with the conditions under which it was slaughtered. To minimize variations the preparation of meat media requires extensive quality control, and this may be impracticable for small laboratories. In these circumstances, commercial meat extracts or dehydrated meat media are often more convenient. When meat media are to be used as the basis for fermentation studies, they should be tested for the absence of fermentable carbohydrate. 2.2.4 Meat extract. Commercial meat extracts contain soluble organic bases, protein degradation products, vitamins and minerals. As these extracts are readily available and easy to use they have largely superseded fresh meat infusions, which are both time-consuming to prepare and variable in quality. 2.2.5 Yeast extract is made from bakers' or brewers' yeast and is a rich source of amino acids and vitamins of the B-complex. In culture media it is used to supplement or replace meat extracts. Meat extract (1%) can be replaced by yeast extract (0.3%) in Nutrient Broth without significant change in the growth-promoting capacity. 10

[2.2]

2.2.6 Blood. The choice of blood is often a matter of convenience and may depend on the animals kept by a laboratory. Horse blood from commercial sources is commonly used, but the blood of other species (man, cow, goat, rabbit, sheep) may be necessary for special purposes; they should be free from antimicrobial agents. Sheep Blood Agar can be used for detecting the different haemolysins of staphylococci and streptococci although bovine blood may give stronger reactions; haemolysis of sheep and human blood may be used also in the identification and biotyping of some species of Vibrio. Sodium citrate is inhibitory to staphylococci (Rammell, 1962) as is Liquoid to some anaerobic cocci and Streptobacillus (Holdeman & Moore, 1972). In general, defibrinated horse blood is preferable; it should be relatively fresh and should not be used if haemolysed. Blood must be stored in a refrigerator but should not be allowed to freeze; all blood products must be tested for sterility as well as for inhibitory substances such as citrates. 2.2.7 Plasma is used for demonstrating coagulase activity. In medical bacteriology laboratories, human plasma is usually preferred but rabbit plasma may be used. As some bacteria utilize citrate, oxalated plasma is better but citrated plasma may be used when heparin is added (Harper & Conway, 1948). Plasma may be obtained by removing the supernatant of the blood-anticoagulant mixture after the red cells have settled. Blood samples obtained for biochemical examination and containing sodium fluoride or ethylenediaminetetra-acetic acid (EDTA or 'sequestric acid') are to be avoided. Liquid plasma is an unstable product liable to coagulate or to form particles which cause a turbidity or deposit. It should not be filtered. Plasma should be stored in a refrigerator but should not be frozen. Dehydrated plasma is available commercially. 2.2.8 Serum is prepared from blood, collected without addition of an anticoagulant, by removal of the liquid that separates when the clot contracts. Alternatively it may be obtained from citrated plasma clotted by the addition of calcium. Serum should be sterilized by filtration. Horse serum may contain a maltase and an amylase and it is essential that these be inactivated by heat before addition to maltose- or

MEDIA CONSTITUENTS

starch-containing media (Goldsworthy, Still & Dumaresq, 1938). Hendry (1938) reported that the maltase was inactivated at 75 °C for 30 minutes but not at 55 °C for 4 hours; Goldsworthy, Still & Dumaresq (1938) recommended heating serum at 65 °C for 1 hour. Horse serum kept at 0 - 4 °C for a month or longer may show a deposit, believed to contain calcium, lipids and protein (Roche & Marquet, 1935) and this deposit may be mistaken for bacterial contamination. 2.2.9 Ascitic and hydrocele fluids are preferred to serum by some workers in hospitals. Most laboratories do not use them because they are not generally available. 2.2.10 Bile contains several bile acids as compounds conjugated with amino acids; bile acids can also form addition compounds with higher fatty acids and other substances. Bile also contains the pigments bilirubin and biliverdin. Fresh ox bile (ox gall) has been superseded by bile extract, dehydrated bile or bile salts. Bile extract, a dark yellowish-green material, is prepared by concentration of fresh bile, extraction with 90% ethanol, and evaporation of the ethanolic extract. A 10% solution of the dehydrated product is equivalent to fresh bile. 2.2.11 Bile salts. Commercial bile salts are prepared by extracting dried ox or pig bile with ethanol, decolorizing the extract with charcoal, and precipitating the bile salts with ether to form a water-soluble yellowish-brown hygroscopic powder. When prepared from ox bile the salts consist mainly of sodium taurocholate and sodium glycocholate with smaller amounts of the sodium salts of taurodeoxycholic and glycodeoxycholic acids. The bile acid conjugates may be hydrolysed by alkali, and it is possible to prepare sodium cholate or deoxycholate in this way, but these substances are not chemically pure. Several workers (Mair, 1917; Downie, Stent & White, 1931) showed that, in selective culture media, deoxycholic acid is the most active component of bile; its effects were studied by Leifson (1935). 2.2.12 Gelatin is the protein obtained by extraction of collagenous material from animal tissues, and is available as sheets, shreds, granules or powder. A

[2.2] gelatin of pharmaceutical or edible quality should be used for culture media. When immersed in water below 20 °C, gelatin does not dissolve but swells and imbibes 5-10 times its volume of water. Solution is effected by heating and the solution gels on cooling to about 25 °C. Gelatin has little nutritive value but is used in culture media as a substrate for detecting gelatinase activity. As with agar gels, excessive heating is detrimental and destroys the setting properties. 2.2.13 Carbohydrates. Carbohydrates, collectively called 'sugars', are usually used to enrich media to promote growth or pigmentation, and to determine whether organisms can produce acid or acid and gas from them. The carbohydrates generally used are listed in Table A2 (Appendix A), which also includes glycosides and polyhydric alcohols, with concentrations of aqueous solutions suitable for addition to media. The concentration of carbohydrate in oxidation and fermentation studies is usually 0.5-1%; 1% carbohydrate is preferable as reversion of the reaction is then less likely. Some carbohydrate solutions may be sterilized by autoclaving whereas with others decomposition may occur. Durham (1898) recommended steaming for 'sugars' but Mudge (1917) found that maltose and lactose suffered greater hydrolysis when steamed for 30 minutes on each of three days than when autoclaved at 121 °C for 15 minutes. Smith (1932) showed the adverse effect of heat and the accelerated decomposition of glucose and maltose in the presence of phosphate. Although Whittenbury (1963) found that some lactic acid bacteria differed in oxygen requirements for fermentation in media (i) made up with carbohydrate before sterilization, and (ii) with filtered or autoclaved carbohydrate solutions added to previously autoclaved basal medium, he could not detect any significant difference in the ability of the organisms to utilize the substrate sterilized by the different methods. Such sugar solutions are often sterilized by momentary autoclaving or by steaming but for most purposes it is better to sterilize them by filtration. We have deliberately omitted dextrin from the list of carbohydrates. White dextrin is generally prepared by heating starch moistened with a small volume of dilute nitric acid and dried at 100-120 °C; it contains up to 15% of starch, the remainder consisting of erythrodextrin. Inferior grades are prepared by roasting 11

CULTURE MEDIA: CONSTITUENTS AND STERILIZATION

starch without acid at 150-250 °C, and have a yellow colour; they are hydrolysed to a greater extent than white dextrin and may contain appreciable quantities of maltose. Because of its variable composition, we do not recommend the use of dextrin. Soluble starch is prepared by treating potato starch with dilute hydrochloric acid until, after washing, it forms a limpid, almost clear solution in hot water; it is insoluble in cold water. 2.2.14 Defined media for studies of bacterial nutrition and carbon source utilization (CSU) tests comprise solutions of mineral salts to which the substrate to be tested is added. For nutritionally exacting organisms, as for example some coryneforms, media such as Mineral Salts Medium E (Owens & Keddie, 1969) may need to be supplemented with 0.02% yeast extract and 2 \ig vitamin B12 per litre. 2.3

Indicators

Indicators are incorporated in some culture media to give visual evidence of pH or other changes occurring during the growth of bacteria. Indicators for this purpose must be non-toxic in the concentrations used; for example, some batches of neutral red may inhibit growth of Escherichia coli in MacConkey Broth or Agar (Childs & Allen, 1953). With some bacteria (e.g. Actinomyces israelii), media containing an ethanolic solution of indicator may give better growth than media with aqueous indicator, thus suggesting that the solvent is used as a more readily available carbon source. Where the indicator is prepared as its sodium salt it is preferable to use water as the solvent. Table Al of Appendix A lists the pH indicators commonly used; the amounts recommended are for the addition of concentrated indicator solutions to culture media and are not necessarily suitable for the colorimetric determination of pH values. Indicators of oxidation-reduction potential (redox indicators) have limited use in culture media. Examples are methylene blue or resazurin in thioglycollate media and methylene blue in milk (Ulrich, 1944). The use of tetrazolium compounds such as TTC (2,3,5-triphenyltetrazolium chloride) as indicators of bacterial growth has been advocated, for example, in motility media (Kelly & Fulton, 1953) and in KCN Broth (Gershman, 1961). With bacterial growth, the 12

[2.2]

colourless reagent is reduced to an insoluble red formazan. 2.4

Sterilization

Sterility implies freedom from all viable microorganisms including spores so that the term sterilization is strictly a misnomer in relation to the processes to which media or their constituents are subjected. For example, the heat applied during steaming is sufficient to kill only vegetative bacteria; media that appear to be sterile may therefore still contain viable spores of thermophiles that do not grow at the temperatures at which 'sterility' tests are usually carried out (37 °C); and labile constituents of media that are normally kept at about 4 °C may contain organisms that can grow at room temperature but not at 37 °C. Even autoclaving does not provide an absolute guarantee of sterility as the exponential nature of the survival curve means that, during heating, the proportion of organisms surviving per unit of time is constant. The microbiological concept of sterility, however, is well understood and we do not therefore intend to define or qualify the term 'sterilization' other than to say that for culture media the particular method chosen should take account of (i) the likely initial bacterial (and spore) concentration, (ii) the confidence level required for 'sterility' of the final product, and (iii) the need to preserve heat-sensitive constituents. In contrast, for reasons of laboratory safety, all cultures for discard (including those from environmental samples), as well as infected glassware and plastic containers must be sterilized in the strict sense of the term even though some of the material will ultimately be destroyed by incineration. The use of chemical disinfectants for the disposal of cultures and infected materials should not be permitted; it should be an inviolable rule that all discarded cultures are sterilized by autoclaving. The lethal action of heat on bacteria depends on both the temperature and the time for which it is applied: the higher the temperature, the shorter the time needed. Sporing bacteria are more resistant to heat than vegetative bacteria but all sporing forms, including different strains of the same species as well as different organisms of the same strain, are not equally resistant (Stumbo, 1973). Some, such as the spores of Bacillus subtilis, are killed by a short expo-

[2.4]

STERILIZATION

sure to 100 °C; others, such as those of B. stearothermophilus, can resist boiling for hours or survive conventional laboratory autoclaving for short times and for this reason have been used as indicators of the efficacy of steam (though not dry-heat) sterilization. To ensure adequate heat-penetration and the destruction of discarded culture, autoclaving must be maintained either for longer times or at temperatures higher than those used for sterilizing culture media. In practice, for sterilization of discarded cultures we use a time-temperature combination of 20 minutes at 121 °C (1.06 kgf/cm2). Alternatively, 10 minutes at 126 °C (1.41 kgf/cm2) as sensed within the load is satisfactory for modern autoclaves with 'make-safe' cycles.

Table 2.1 Relation between temperature and pressure of saturated steam at some commonly used autoclaving temperatures

2.4.1 Autoclaving. An autoclave is a pressure vessel which must be regarded as a potential hazard and protective clothing should be worn when opening it for unloading. During autoclaving, a temperature above 100 °C is achieved with steam under pressure, the latent heat released in condensation rapidly heating the load to the temperature of the surrounding steam. When all the air has been expelled and the autoclave chamber is filled with saturated steam there is a direct relationship between temperature and pressure (Table 2.1); if any air is present, the temperature will often be lower than that corresponding to the steam pressure. Overheating is detrimental to most culture media, but autoclaving is the most satisfactory method of sterilizing material or media that will withstand temperatures over 100 °C, the usual temperature-time combinations for most microbiological media being either 115 °C (0.69 kg/cm2 ) for 20 minutes or 121 °C (1.06 kg/cm2) for 15 minutes. As the rate of heat penetration into large containers is slow, especially if they contain agar, small volumes (less than 1 litre) are preferable; when the volume does exceed 1 litre, the time but not the temperature should be increased to aid heat penetration. Containers such as test tubes, flasks and bottles should be of a capacity sufficient to allow a generous head space; containers of Stuart's (1959) transport medium, which should be filled completely, are an exception. For consistent autoclave results, the temperatures within media in standard loads should be established and the loading patterns adhered to strictly. The temperature inside

* 14.7 lbf/in2 at sea level. NOTE: 1 lbf/in:2 = 0.0689 bar == 0.07 kgf/cm2

Temperature

Pressure above standard atmosphere*

°C

bar

kgf/cm2

lbf/in2

100 105 110 115 120 121 125 126 130 134

0 0.21 0.43 0.69 0.98 1.05 1.32 1.39 1.70 2.04

0 0.20 0.43 0.69 0.99 1.06 1.33 1.41 1.72 2.10

0 3.1 6.2 10.0 14.2 15.2 19.1 20.2 24.7 29.6

the autoclave should be allowed to fall to 100 °C before it is vented to the atmosphere and then to 80 °C or below before the door is opened to remove the contents. The detrimental effects of heat on media begin at about 60 °C and include the decomposition of growth factors; caramelization of sugars; the Maillard reaction between sugars and amino compounds; and pH changes (Peer, 1971). As the lethal effect of heat on bacterial spores increases about tenfold compared with twofold for chemical effects with each rise of 10 °C in temperature, a higher temperature for a shorter time should cause fewer chemical changes in media than a lower temperature for a longer time. Similarly, during cooling, the chemical effects reduce twofold and the lethal effect on spores tenfold for each fall of 10 °C in temperature. These effects thus emphasize the importance of minimizing the heating and cooling times of the autoclave cycle; if possible, steam should be admitted at a high temperature for rapid sterilization and the autoclave cooled to below 80 °C as quickly as possible. Media in small containers (less than 1 litre) will contribute to the speed of heating and cooling (Everall & Morris, 1975). 2.4.2 Momentary autoclaving. A high-temperature, short time (HTST) procedure represents a compromise between sufficient reduction in the number 13

CULTURE MEDIA: CONSTITUENTS AND STERILIZATION

of microorganisms present and the limitation of undesirable side-effects. The heat or steam is turned off as soon as the autoclave reaches the required temperature (e.g. 121 °C) chosen from experience with known media and uniform loads. The valve is opened to vent the chamber to the atmosphere when the temperature in the bottles falls to 100 °C, and the autoclave is unloaded below 80 °C. 2.4.3 Steaming, including Tyndallization, consists of exposing the medium to the vapour of boiling water in a non-pressurized vessel, though the medium itself seldom reaches 100 °C. Sterilization by steaming may be carried out once only, or on three successive days, when it is a high temperature form of fractional sterilization known as 'Tyndallization'. Sterilization by boiling or steaming may be necessary when media cannot be autoclaved without detriment to their constituents, for example those containing selenite or tetrathionate which are sometimes referred to as 'self-sterilizing'. Many of those that cannot be sterilized by autoclaving are enrichment or selective media used for isolating particular organisms from a mixed flora; in these circumstances, sterility is not always essential. Tyndallization has limited use in the media department and is suitable only for nutrient media in which spores, if present, can germinate during the intervals between successive steaming. It was originally used for Litmus Milk medium, which was heated at 80 °C for one hour on three successive days. It is a time consuming procedure which has been superseded by autoclaving at 115 °C for 10 minutes. 2.4.4 Inspissation is a fractional sterilization procedure carried out at a temperature sufficiently high to coagulate serum or other heat-labile constituents such as egg-white and it consists of heating them at 75-80 °C for one hour on three successive days. An alternative method of inspissation using older autoclaves is described for Dorset Egg medium in Appendix A, but it is not suitable for modern autoclaves fitted with temperature-sensed door-release mechanisms. Other methods for inspissation were described by Levin (1943), Foster & Cohn (1945), Spray & Johnson (1946) and Brown (1959).

14

[2.4]

2.4.5 Other methods of 'heat 9 sterilization. These include the use of dry heat, ultra-violet light, microwave ovens and gamma irradiation, though they each have practical limitations for the sterilization of media. For apparatus and glassware, sterilization in the strict sense can be achieved by dry heat in a hot air oven provided the temperature is raised to and maintained at 160 °C for at least one hour; a higher temperature will char paper and cotton wool. 2.4.6

Sterilization by filtration

Sterilization by filtration has the advantage that it is a process suitable for solutions of thermolabile materials; its disadvantages include the possibility of hidden defects in the filtration apparatus, the need for sterilization of the apparatus before use, the possibility of selective adsorption from dilute solutions and of pH changes, and difficulty in cleaning certain filters after use. Filtration is not just a mechanical sieve-like action depending on porosity and thickness of the filter but is also a complex physico-chemical procedure involving the ionic charge on the filter and the pH of the solution. Bacterial filters may be made of porous porcelain, kieselguhr (diatomite), sintered glass, or cellulose esters. Doulton and Pasteur-Chamberland candles or cylinders are made of porous porcelain and are available in varying porosities, not all of which are suitable for bacterial filtration. Berkefeld and Mandler filters are made from kieselguhr. Porcelain and kieselguhr filters carry a negative charge. Sintered or frittered glass filters are available in varying porosities, no. 5 being suitable for bacterial filtration; it is usually available as a 5/3 combination consisting of a no. 5 filter supported on a no. 3 filter for mechanical strength. Sintered metal filters are not widely used in bacteriology as they are not completely inert to the action of materials likely to be found in culture media. Collodion and asbestos filters have largely been replaced by the more convenient membrane filters composed of cellulose esters, nylon or polytetrafluoroethylene (PTFE), which are stored in the dry state; although cellulose filters are somewhat brittle, they all have good wet strength and may be sterilized by autoclaving.

Principles of isolation

3.1

Isolation methods

Isolation begins with the collection of the specimen. Normally the clinician takes the specimen and sends it to the laboratory, but there are occasions when the bacteriologist should go to the patient or vice versa, so that fresh material can be examined while 'hot*.

3.1.1

Direct microscopy. Since amoebic and

bacillary dysentery cannot be distinguished clinically, it is essential when amoebic dysentery is endemic or is suspected, to examine a freshly passed stool on a warmed microscope stage to see the characteristic movements of vegetative Entamoeba histolytica; even better specimens may be obtained at sigmoidoscopy. Only by seeing the movement of E. histolytica can it be distinguished from Entamoeba coli; the differences between the encysted forms are not sufficiently great or constant to be diagnostic. The serous fluid of a primary chancre collected in a capillary tube can also be examined unstained by microscopy for spirochaetes. Stained material from leprosy lesions usually shows abundant organisms which, as yet, defy the usual cultural methods. It is usually unrewarding to stain blood films for bacteria, but in hot countries bacteria are not the only causes of fever; malaria parasites and other bloodborne protozoa should therefore always be sought. Pus, cerebrospinal fluid (centrifuged deposit), pleural effusions, and other transudates may show bacteria or other microorganisms when stained by Gram's method; if none is seen, a film stained by the ZiehlNeelsen (ZN) method may reveal acid-fast rods. Failure to find bacteria is not conclusive and, like other negative results, may indicate simply that the method used was not sensitive enough; Corper (1928) estimated that 100 000 acid-fast bacilli per millilitre

of sputum was the minimum concentration that could be detected by direct microscopy. However Tazir et al. (1979) suggested that 90-96% of specimens containing between 30 000 and 50 000 acid-fast bacilli per millilitre and 5 0 - 5 8 % of those containing between 2 000 and 5 000 bacilli per millilitre give positive Ziehl-Neelsen stained smears. A negative microscopic result could indicate that the infecting agent was a virus or a mycoplasma. Urethral smears, on the other hand, may be more successful than cultures, and after seeing Gram-negative diplococci, treatment can be started before the results of culture are available. When treatment is started before the specimen is taken, cultures are unlikely to yield growth of gonococci. To summarize: microscopic examination of all specimens except blood (in temperate climates), faeces, and rectal swabs are always worth making but prolonged and exhaustive microscopic examination for tubercle bacilli does not justify the effort expended on them. 3.1.2 Fluorescent microscopy. The use of fluorescent and immunofluorescent techniques for the detection of particular organisms or bacterial antigens has increased rapidly and greatly improved the chances of early diagnosis. These methods are outside the scope of this Manual and the reader should consult, for example, Fluorescent Protein Tracing by Nairn (1976). 3.1.3 Cultural methods. With knowledge of the site of origin of the specimen and from examination of stained smears, the clinical bacteriologist is able to anticipate the kinds of organism likely to be present so that the culture media can be matched to the 15

PRINCIPLES OF ISOLATION

organism(s) expected. Knowledge of the nature of likely contaminating and therefore unwanted organisms is also essential, although it is often true that what is looked for is isolated but the unexpected or unsought organism is missed. Anaerobes should not be regarded as special cases and should be sought in most specimens. In considering isolation procedures the strategy must start from the nature of the specimen; it is important to know whether it is likely to contain only one kind of organism as in cerebrospinal fluid; several significant species as in many anaerobic infections; or whether, as in material from the oropharynx, gastrointestinal tract and lower female genital tract, the specimen will yield a background of bacteria from which the relevant pathogens have to be distinguished and separated. Specimens from blood and tissues that are normally sterile are inoculated on rich, non-inhibitory media, to isolate all kinds of organisms present. And bearing in mind that not all bacteria grow in air, replicate plates should be made for incubation with added CO2 and under anaerobic conditions. The specialized techniques used for isolating anaerobes are described in detail in several monographs which give useful laboratory tips based on years of experience (Holdeman, Cato & Moore, 1977; Willis, 1977; Sutler, Citron & Finegold, 1980; Willis & Phillips, 1983, 1988). In clinical laboratories anaerobic jars provide the usual means of obtaining an oxygen-free atmosphere for the culture of obligate anaerobes. Modern anaerobic jars operate by catalytic removal of oxygen from the internal atmosphere by palladium in the presence of excess hydrogen. A variety of jars is available commercially for both evacuation-replacement methods and internal gas generation. Anaerobic bacteria vary widely in their sensitivity to oxygen and the choice of technique depends to some extent on the aerotolerance of the species sought and on the numbers of organisms present. Many of the anaerobes commonly implicated in human infections are not extremely oxygen-sensitive and are often present in clinical specimens in predominant numbers. Thus, provided careful attention is paid to operational techniques, the anaerobic jar can be relied upon for the isolation of most anaerobic species of clinical significance. An anaerobic cabinet, equipped with glove ports 16

[3.1] and a rigid airlock for transfer of materials, provides an oxygen-free environment in which conventional bacteriological techniques can be applied to the isolation and manipulation of obligate anaerobes in conditions of strict and continuous anaerobiosis. Such a continuity of anaerobiosis is intrinsically more efficient than the cyclic operation of multiple anaerobic jars. A few organisms, notably some of the pathogenic mycobacteria, grow slowly and are best cultivated in screw-capped containers to prevent the medium drying out. Mycobacterium leprae does not grow on culture media and knowledge of its presence in tissues depends on microscopical evidence. The leprosy organism will grow in the footpad of the mouse or the nine-banded armadillo; the latter has provided the material for a vaccine. Although bacteriological culture methods will not isolate viruses, Mycoplasma species in specimens will grow from time to time on media containing blood or serum in a closed - but not necessarily anaerobic - jar. When the presence of mycoplasmas is suspected, special media (the so-called PPLO media) should be used for their isolation and propagation; such media are available commercially. Specimens from areas or sites that have a normal microbial flora cannot be treated so cavalierly. Look at the specimen with an appreciative eye, for it must be inoculated onto appropriate selective media; for example, a loose watery stool and faeces with flecks of blood and mucus should be inoculated onto a number of different media. Experience is the best guide but in its absence several selective media should be used, including the less inhibitory media such as MacConkey or Eosin Methylene Blue (EMB) Agar for faeces from adults and children; for stools from babies use Blood Agar on which the enteropathogenic strains of Escherichia coli can be successfully isolated and identified. For enrichment, a large inoculum of the specimen (usually faeces) is placed in about 10 ml of medium such as Tetrathionate or Selenite Broth; after incubation for a few hours subcultures are made to selective (inhibitory) media such as Deoxycholate Citrate Agar (DCA) or Salmonella-Shigella (SS) and to basal or only slightly inhibitory media (MacConkey or EMB Agar). After incubation overnight the plates are examined and colonies selected for subculture to non-

[3.1]

ISOLATION METHODS

inhibitory media. This selection of colonies is not the end of the isolation process as further plating on noninhibitory media may be necessary to obtain a pure culture suitable for characterization tests. In the absence of good growth on the first plates subcultured from the enrichment medium, further subcultures are made after longer incubation. Enrichment media such as Tetrathionate Broth can alsobe used as transport media for faeces. A few points need special attention. (i) Blood should usually be inoculated into large volumes (at least ten times that of the specimen) of broth containing 0.03 to 0.05% sodium polyanethol sulphonate (Liquoid) or other anticoagulant; incubation should be continued for at least 7-10 days and the broth subcultured at intervals to detect growth. Liquoid may inhibit growth of some anaerobic cocci; Holdeman & Moore (1972) recommend that blood cultures should be inoculated into media with and without Liquoid. (ii) On first isolation most bacteria grow better in an atmosphere containing 5-10% CO 2 but CO 2 inhibited mutants of Escherichia coli and Salmonella typhimurium do occur occasionally (Roberts & Charles, 1970). CO2 is either essential or stimulatory for the growth of many anaerobes and is a well-established supplement to an anaerobic atmosphere. A precise concentration is probably not critical for growth but may be important when a controlled atmosphere is required, as for example in antibiotic sensitivity tests. (iii) Specimens from patients with suspected gonorrhoea or meningitis should be inoculated on to freshly poured (and undried) Chocolate and Blood Agar plates; when the inoculation of plates cannot be made speedily, the swab or specimen should be put into transport medium (Stuart, 1959). (iv) Some pathogenic mycobacteria grow slowly and usually not on the standard media used for pyogenic pathogens. Lowenstein-Jensen Egg medium is widely used for the growth of mycobacteria but in some countries Middlebrook Agar (Difco) is preferred. The growth of Mycobacterium bovis is enhanced by the presence of sodium pyruvate (Stonebrink, 1961) and M. johnei requires an iron chelating agent, mycobactin, which can be supplied by incorporating killed M. phlei in the medium (Francis et al., 1953). As incubation will continue for

several weeks the medium should be in screw-capped containers and have plenty of water of condensation (synaeresis). Specimens containing mycobacteria often have a mixed bacterial flora; to isolate the more slowly growing mycobacteria the specimen must first be treated (with acid or alkali, to which mycobacteria are usually resistant) to destroy the more rapidly growing, and less acid- or alkali-resistant, bacteria. After treatment for a short time (usually about fifteen minutes) the acid or alkali can be neutralized before the specimen is inoculated onto appropriate media.

3.2

Importance of pure cultures

Many difficulties in identification are due to the use of an impure culture as starting material. Before an organism can be identified it must be obtained in what we glibly describe as a 'pure culture'. By this we mean the descendants of a single colony obtained after plating the material in such a way that much of the growth consists of well-isolated colonies; it is only an assumption that these have developed from a single organism or a single clump of similar organisms. In routine diagnostic bacteriology a single plating may have to suffice but replating can always be made with advantage, and, as explained below, is essential when highly selective and inhibitory media are used for the primary culture. A 'pure culture' is one that generally breeds true; this means that when replated, the majority of the daughter colonies will be like the parent, though occasionally (perhaps once in several million times) a bacterial cell will mutate and the colony developing from it will consist of organisms that have changed a character. A 'pure culture' retains its original characters because the chances are, on an ordinary nutrient medium, millions to one against subculturing the mutant, and also because once satisfied that the culture is pure we no longer choose isolated colonies (lest, perchance, the mutant is picked) but subculture from a sweep (or pool) of several colonies. When two organisms grow together as in an impure culture, one of four things may happen: (i) each organism may grow independently; (ii) one may produce a substance that will enable the other to grow or grow better in the particular medium (synergy); 17

PRINCIPLES OF ISOLATION

(iii) one may produce a substance (bacteriocin) that inhibits the growth of the other (antagonism); or (iv) one may grow faster than the other and deprive the second of some essential part of its food supply. In (i) and (ii) characterization of the impure culture would probably yield a summation of characters unless for example one organism produces acid and the other an equivalent amount of alkali to neutralize it. In (iii) and (iv) the characterization will be that of the organism which grows at the expense of the other. In (i) and (ii) an organism A (characterized as X+, Y-, Z-) growing with B (X-, Y+, Z-) in mixed culture (A+B), might be characterized as X+, Y+, Z-, which might be characteristic of a third organism C, and the mixture would inevitably be misidentified. Often bacteriologists look for a particular organism, such as a shigella in faeces or Corynebacterium diphtheriae in a throat swab, and isolations are usually made on selective or differential media which contain substances that inhibit the growth of some (unwanted) organisms. The inhibitory substance(s) does not kill the unwanted organism but merely suppresses or retards its growth on that medium; when an unwanted organism inhibited in this way forms part of a colony made up mainly of the suspected pathogen, subculture to another medium will result either in further suppression of growth, or, in the absence of the inhibitory substance, a resumption of growth in competition with the pathogen sought. From an inoculum of faeces on Deoxycholate Citrate Agar medium a colourless colony (indicating a lactose non-fermenter) is likely to be made up of lactose non-fermenters and a few suppressed lactose fermenters. Subculture to Lactose Peptone Water will allow the lactose fermenters to grow and produce acid from the lactose and, unless the colony is replated on a non-inhibitory medium, the presence of the lactose non-fermenters may be overlooked. A working routine should be developed so that, before a culture is assumed to be pure, colonies from a selective medium are replated on a noninhibitory and preferably differential medium. Until a culture is known to be pure, it is a waste of time to attempt any characterization tests. The reader may think that we have laboured the presence of mixed colonies, but experience has shown that many of the cultures difficult to identify are, in fact, mixed and came from colonies on inhibitory media. We cannot stress too strongly the 18

[3.2] importance of obtaining a pure culture before attempting to identify it. While inhibitory media are the main source of impure cultures, other causes are sufficiently common to warrant mention here. The presence of a 'spreader' on a plate may be difficult to detect; this spreading growth will also cover any discrete colonies on the plate. The most troublesome spreading organisms are members of two genera, Proteus and Clostridium, and different methods must be used to purify cultures contaminated by them. Proteus species grow readily on most media but swarming can be inhibited by bile salts and by substances in some of the selective and inhibitory media on which Proteus organisms produce discrete colonies. Thus, a culture contaminated by a Proteus species can be plated on MacConkey Agar; if, however, the organism sought will not grow on this medium, the contaminated culture should be spread on plates of a richer nutrient medium in which the agar concentration has been increased to 7% (Hayward & Miles, 1943). It is much more difficult to purify a culture contaminated by a Clostridium species. This difficulty of freeing a culture from a clostridial contaminant applies also to separating one Clostridium species from another and explains why it takes so long to identify many of them. Since more than one species of this genus may occur in the same material, it is not surprising that the original descriptions of many socalled species of Clostridium were based on mixtures of two or more species. The separation of Clostridium species may entail a long series of platings and selection of colonies unless one of the mixture is a pathogen with invasive properties. Such an organism might be isolated from a remote site (e.g. the surface of the liver) when an animal dies after subcutaneous or intramuscular inoculation of the mixed culture in a hind limb. Separation may also be achieved when the spores of the strains in the mixture differ in their resistance to heat or when one of the strains is motile and the other non-motile. The separation of two pathogens may depend on the ability of the bacteriologist to recognize which species (or even which toxin types) are present. Much information can be obtained from in vitro tests, such as lecithinase production on EggYolk medium, before going on to in vivo tests in animals.

IMPORTANCE OF PURE CULTURES The contamination of bacterial cultures by Mycoplasma species (pleuro-pneumonia-like organisms) is not believed to be a serious hazard, but our appreciation of these organisms is much less than that of ordinary bacteria, and their presence may escape detection. Mycoplasmas grow slowly and generally only on media enriched with blood or serum, and if they are initially present as contaminants they are likely to be outgrown by the more hardy and less fastidious bacteria. However, they should be borne in mind and their presence suspected if bacterial cultures show inconsistencies on repeated testing. Bacterial cultures that have been isolated on media containing antibiotics or from patients treated with antibiotics, may show ' g \ ' L ' or other aberrant forms, which may be confused with mycoplasmas because they grow slowly and produce very small colonies.

3.3

Screening tests

Screening tests tejke various forms and, as improvements are constantly being made, only an outline of their development is given here together with a few references for those who propose to use these diagnostic aids. As the main objective of screening tests is economy of time and material, we start with a statement applicable to all, that, for quick results, heavy inoculation is essential. The tests are of two kinds: (i) eliminating, those intended to identify unwanted and (believed to be) unimportant organisms; and (ii) presumptive, those tests that, with media containing several substrates (multitest media), will indicate to which major group an organism probably belongs. Both kinds of screening tests rely heavily on the assumption that the test organism is in pure culture, and this assumption must be made at a time in the isolation and identification programme when the odds are against it. Eliminating tests are usually quite simple as, for example, the inoculation of urea medium when looking for salmonellas or shigellas; if the urea is hydrolysed that particular culture can be discarded, for it would be a most unusual salmonella or shigella that produced urease. Knox (1949) elaborated on this principle by placing coverslips (to detect gas production) and impregnated test papers on the surface of a

[3.2] plate of multitest medium inoculated with a test organism; from the plate, it was possible to detect H2S production, mannitol, sucrose, and lactose fermentation, and swarming of the organism. In conjunction with a tube of urea medium the tests covered a wide range; some of the tests, when positive, suggested that the organism was unlikely to be a pathogen and that the culture could be discarded. Lanyi & Adam (1960) combined the impregnated paper discs of the Knox plate with a selective basal medium so that colonies from the primary plate could be used as inocula; deoxycholate in the medium prevented interference by many organisms likely to be present in cultures of faeces. The elimination of any culture carries the risk of loss of a rarely encountered organism or one that may be an unrecognized pathogen; this is the penalty for discarding primary plates before investigations are completed. The moral is never to discard a specimen or primary plate until all tests have been completed and the identification made satisfactorily. This may seem a counsel of perfection but most of us learn it by bitter experience; and it did lead to the discovery, among other things, of penicillin by Fleming (1922). Presumptive identifications can be made by inoculating media containing several substrates and one or more indicator(s). Some of these media contain inhibitory substances so that a colony from a selective medium can be used as inoculum, but most multitest media require the inoculum to be a pure culture. By a judicious choice of indicators and substrates, multitest media may show a sufficient range of colour or other changes to make a preliminary allocation of the test organism to a major group or subdivision. Multitest media and methods only justify themselves when they are both quick and accurate so that effort can be concentrated on cultures showing the reactions of established pathogens; to use multitest media to make the final identification is indefensible. But by eliminating unwanted organisms, workers in busy laboratories should have time to investigate the presumptive pathogens by the more informative and reliable tests described in Chapter 4. Because lactose fermentation was believed to be of paramount importance, Russell (1911) put ten times more lactose than glucose in his double-sugar medium. Kligler (1917, 1918) introduced lead acetate to the medium, which was blackened when sufficient 19

[3.3]

PRINCIPLES OF ISOLATION

H2S was produced. In another variation, substituting mannitol for lactose, Kligler & Defandorf (1918) claimed to be able to distinguish the Shiga from the Flexner dysentery bacillus. To increase the differentiating power still further, Kendal & Ryan (1919) experimented with various combinations of sugars and ended with two double-sugar tubes which included glucose, lactose, sucrose, and mannitol. Other combinations and the addition of other substrates to bring in more characters have followed, and another two-tube test (Kohn, 1954 as modified by Gillies, 1956) became popular for the screening and presumptive identification of intestinal pathogens. Kligler's Iron Agar (1917, 1918) formed the basis of TSI (Triple Sugar Iron Agar) which was apparently developed simultaneously but independently by Sulkin & Willet (1940) and by workers in the Difco Laboratories {Difco Manual, 1953). Although TSI was introduced as a multitest medium it has become an unofficial standard for H2S production at the low degree of sensitivity that has differential value among enteric bacteria. Screw-capped containers, which do not allow volatile products to escape and so ittay affect the colour of indicators, are not particularly suitable for multitest media (Marcus & Greaves, 1950). Most multitest sloped media are intended for use in the identification of enterobacteria; others are aimed at detecting pathogenic vibrios (Hsu, Liu & Liao, 1964), and Chapman (1946, 1952) described some for identifying staphylococci. Multitest media have been exploited commercially especially for the identification of enterobacteria, which are not only common and widely distributed but are easily identified and placed in their major groups by simple biochemical tests. Many schemes or systems have been marketed in the USA, Europe and the Far East, though few have had more than a fleeting popularity. Identification methods that rely heavily on prelimi-

20

nary screening on multitest media should not be used; it is far better to work with pure cultures on agar slopes and use them to inoculate all the media needed to make an identification. There are many disadvantages in multitest media, not least the possible interaction of one chemical reaction with another; acid produced from a fermentable sugar may inhibit the blackening of the iron indicator in TSI medium (Bulmash, Fulton & Jiron, 1965), and ammonia produced from peptone may inhibit urease production (Stewart, 1965). There may also be interference in biochemical reactions, as nitrite interferes with the detection of indole when tested by some of the older methods (Smith, Rogers & Bettge, 1972). 3.4

Rapid tests

In the previous edition of this Manual, single substrate and rapid tests which could be used for screening were described. Clarke & Cowan (1952) developed a series of micromethods in which constitutive and induced enzymes in heavy suspensions of bacteria acted on the test substrate in a buffered solution. They believed, together with Manclark & Pickett (1961), that bacterial characterizing tests should be carried out as far as possible in such a way so that the organism acted on only one substrate in each test. This view has since been confirmed by the many different rapid methods which have been marketed commercially since the API system was first described by Buissiere & Nardon in 1968. Despite some early discrepancies with conventional test results (Smith, Rogers & Bettge, 1972), these rapid characterization tests have now become an integral part of the routine in many laboratories, either as short sets for screening purposes, as with enterobacteria, or as extensive sets for species identification, as with the genus Bacillus. In view of their importance and widespread use, we discuss them more fully in Section 4.6.

Bacterial characters and characterization

Features such as brightly coloured pigments may be characteristic of a few species as well as a good pointer to the nature of the organism and its ultimate identification. But pigments may mislead, as in the Biblical case of bleeding polenta which is no longer considered to be a miracle but a phenomenon that can be produced experimentally by a bacterium and in nature is commonly produced by a yeast (Merlino, 1924; Gaughran, 1969; Cowan, 1956a, 1910b). As bacteria present few gross diagnostic features they must be looked at closely; the characters sought and the tests applied will depend on knowledge and expertise as well as experience with similar organisms; the approach to identification will be conditioned by professional training and intuitive skill. The observations made and the tests applied are aimed at characterizing the organism so that it can be described (the technical term for the list of characters is a description) and compared with descriptions of other, previously identified and classified organisms.

4.1

Bacterial characterization

The difference between characterization for classification and for identification lies not so much in the tests themselves as in the emphasis placed on the results of the tests. Although it is not universally accepted, most taxonomists now support the Adansonian concept that, for classification, equal weight should be given to each character or feature. The relationships between strains can be calculated and expressed as similarity either of positive characters (Sneath, 1957/?) or by taking account of both positive and negative features (Hill et al., 1961; Floodgate, 1962; Lockhart & Hartman, 1963); the results of such comparisons can be analysed labori-

ously by making a large number of calculations, or more easily by letting a computer do the hard work (Sneath, 1978). We accept the Adansonian concept for classification, but for identification we attach much weight to some characters, regarding them as having great distinguishing value; we give less weight to others, and no weight at all to some features. Excessive weighting is given to coagulase production by staphylococci; heavy weighting is placed on the urease and phenylalanine deaminase systems in identifying Proteus and Providencia species, and on urease in distinguishing between Alcaligenes faecalis and Bordetella bronchiseptica. Little emphasis is placed on gelatin hydrolysis or liquefaction by staphylococci or micrococci but more weight is given to the same test among the enteric bacteria or the pseudomonads. The variable weighting attached to these characters is largely based on experience but it is always hoped that the assimilation of data from a wide range of bacteria will, in the future, enable the value of a feature to be expressed in a quantitative manner; this has already been done for the Enterobacteriaceae by Edwards & Ewing (1962, 1972) and more recently by Farmersal. (1985). If everyone had ready access to a computer, an almost unlimited number of characters or features could be used, but with tables we are restricted by memory and limited by our ability to recognize similarities and differences when making multiple mental comparisons simultaneously. Because of these limitations a mechanical aid (named the Determinator) was developed by Cowan & Steel (1960, 1961) and tables suitable for use with it were constructed. The original Determinator was restricted by its size to about twentyfive features, but in the simplified form this limitation was removed and, in theory at least, fifty or more 21

BACTERIAL CHARACTERS AND CHARACTERIZATION features could be included in the tables. Few ever used this device, but the tables, which can be used by simple inspection, led to the development of this Manual and indirectly to a more elaborate mechanical device for the identification of enterobacteria (Olds, 1966, 1970). By selection and weighting the number of features used in the individual tables was restricted, and the identification made in stages. At one time Cowan & Steel intended to make tables with the smallest number of tests essential for identification. In the event each second-stage table includes sufficient detail to provide an adequate, but not exhaustive, characterization.

4.2

Choice of characters

In choosing characters for the tables we prefer those that seem to be most constant and tests that give the most reproducible results. Unfortunately, nearly all tests are influenced by factors that are difficult to control, and it is therefore difficult to specify standard methods. All' we can do is to recommend that the materials used (media and reagents) should be controlled as far as possible (see Appendix A) and that environmental factors, such as temperature and the duration of incubation, should be standardized. Various workers have discussed the choice of characterizing tests and methods, and each has their own preference. Sneath & Johnson (1972) suggested statistical methods by which the influence of test error on the correctness of identification could be measured; they estimated that within one laboratory (where the error is likely to be least) test error will be about 5% and they considered that a test with a laboratory error greater than 10% would not be suitable for taxonomic work. We would emphasize the desirability of keeping to the same method of doing a test so that its idiosyncrasies and difficulties become known, and, within the one laboratory, the results become reasonably comparable. We have made innumerable comparative tests but could seldom say unequivocally that one method was better than all the others. To keep the results reasonably comparable we chose certain methods and these became our laboratory standards. Often the choice of method was a compromise between two, sometimes conflicting, demands: firstly to know the truth, and secondly to be 22

[4.1]

able to distinguish between two otherwise similar organisms. It is essentially a compromise to express qualitatively (as positive or negative) what is really a quantitative reaction. An example is the production of hydrogen sulphide; when an organism is grown in a medium with an adequate sulphydryl content, and a sensitive indicator (lead acetate paper) is used, the ability to produce even small amounts of H2S can be detected, but the method is not discriminatory and the results are useless for distinguishing between those salmonellas that produce much and those that produce only a little H2S. However, with a medium deficient in -SH compounds and with a poor indicator (ferrous chloride), only an organism with great ability to produce H2S is positive in the test. Sometimes a test is carried out by different methods when dealing with different groups of organisms. Again taking H2S production as the example: in the genus Brucella the organisms are grown on a medium rich in -SH compounds and lead acetate papers are changed each day so that the result of the test can be expressed as 'H2S produced on the first two days' or 'from the 1st to the 5th day'. We do not know of any other group of organisms in which this technique is used, and its application to other groups might give information of value. On occasions it is thus essential to indicate the method to be used to obtain the results given in a table.

4.3

Characters not used in the tables

Before describing the characters chosen for use in the tables, we think we should state our reasons for omitting some time-honoured characters and tests. Certain features are not used in the diagnostic tables because they are subjective; for example, the smell of staphylococci growing on agar media is unmistakable but also indescribable. The recognition of the finer shades of pigments is a subjective observation; we try to keep to the primary colours and avoid such indefinite subdivisions as, for example, coral red and sky blue. We do not ususally describe colony morphology as this will vary with the medium on which the organism is grown and, except with bacteria such as Corynebacterium diphtheriae var. gravis, is seldom sufficiently characteristic to have diagnostic value.

CHARACTERS NOT USED IN THE TABLES We do not consider that the descriptions of cultures on agar slopes are worth much, and we rarely pay any attention to the type of growth in broth except to note the presence or absence of a pellicle. In a few cases (e.g. Bacillus anthracis) the type of growth in gelatin stab cultures is characteristic, but the inverted fir-tree growth can only be seen when the gelatin columns are deep and the cultures are incubated at about 22 °C. On the whole we pay little attention to the type of liquefaction of gelatin and are content to record the test as 'gelatin liquefied' (positive) or 'not liquefied' (negative). Thus, in this Manual there are no diagrams of the different shapes, edges, surfaces, and elevations of colonies, or of the shape of liquefaction seen in gelatin stab cultures; the elimination of these relics of early bacteriology makes unnecessary a glossary of descriptive terms that now have but limited use. However, lest we should be accused of too biochemical an approach to classification and identification, we must state our belief that cell morphology and thus microscopy does have an important place in characterization. We do not describe serological techniques since the role of serology in classification and identification lies chiefly in the finer subdivisions used for epidemiological purposes. For those who wish to pursue serological analysis we recommend the monographs by Kauffmann (1954), Edwards & Ewing (1962, 1972), and Ewing (1986). The latter are essentially practical treatises and contain such relevant and important details as the identity of the best strains to use for immunization and absorption. However, as the primary subdivision of the streptococci depends on the serological method of grouping introduced by Lancefield (1933) and as this is used in Table 63b, we describe the methods of preparing extracts of streptococci necessary for this test in Appendix C. We do not describe fluorescence microscopy or fluorescent antibody (FA) methods as they are now subjects in themselves. We suspect however that not all laboratories have the necessary equipment and expertise available to use them. Those who want guidance should consult reviews by Cherry & Moody (1965), Georgala & Boothroyd (1968) and Nairn (1976). 4.3.1 Antibiotic sensitivity. To clinicians, the sensitivity of an infecting organism to antibiotics that

[4.3] can be used therapeutically is clearly important. Hence, antibiotic sensitivity tests have become an important part of the clinical laboratory routine and there is a tendency to regard the results of these tests, expressed in an antibiogram, as valuable characteristics for the identification of an organism. Although it is possible to say in general terms that certain species are sensitive or resistant to a particular antibiotic, sensitivity to antibiotics is not a character that has much diagnostic value among the bacteria of medical interest, and with occasional exceptions (e.g. the resistance of most campylobacters to nalidixic acid), the antibiogram has only a minor part to play in identification work. In the case of obligate anaerobes, however, sensitivity to metronidazole is a valuable diagnostic character which serves to distinguish anaerobes as a group from aerobes and facultative organisms (Tables 6.1 and 7.1). While an unusual antibiogram can point to a misidentification (Abrams, Zierdt & Brown, 1971), the list of sensitivities and resistances of a strain from a treated patient may simply reflect the effectiveness or failure of treatment. And, since strains which become resistant in vivo do not usually change their other characters (Brown & Evans, 1963), the deletion of the antibiogram removes only a highly variable character from consideration when the identification is made. Non-fermenting and non-saccharolytic Gram-negative rods belong to groups of bacteria that have so many negative and so few positive characters that it is tempting to accept the antibiogram as a useful tool, if only as an auxiliary one (Pedersen, Marso & Pickett, 1970; Gilardi, 197 lfc). We think that this has limitations when trying to identify such strains from patients, for the possibility of resistance developing as a result of treatment, or from the influence of transfer and resistance factors (Garrod, Lambert & O'Grady, 1981) must not be overlooked. So, although the antibiogram is important and should be reported to the clinician, it should play only a small part in the preliminary characterization and description necessary for accurate identification of organisms isolated from man and other animals especially if they have been treated with or fed antibiotics. However, sensitivity or resistance to antibacterial agents or antibiotics, especially if they are not used for treatment or included in animal foods, can be helpful in the preliminary screening and differentia23

BACTERIAL CHARACTERS AND CHARACTERIZATION tion of certain organisms or groups of organisms: for example the sensitivity of pseudomonads and many vibrios to the pteridine derivative O/129, the sensitivity of most anaerobes to metronidazole and the resistance of most campylobacters to nalidixic acid.

4.4

Primary tests used in the tables

When the tables for this Manual were first prepared, the Gram reaction was used as the starting point with morphology, fundamental reactions such as ability to grow in air, catalase and oxidase production, and the method of carbohydrate breakdown, for subsequent subdivisions. One advantage of a table is that several characters of different groups can be seen simultaneously and compared, whereas this is not possible with the genealogical type of chart or dichotomous key. We were surprised to find that most bacteria could be placed in a genus or small group of genera by using the results of a limited number of selected tests. 4.4.1 Gram reaction. Gram did not describe a stain but a method in which he used stains and solutions devised by others; to this day its mechanism is not fully understood, but we do know that the Gram reaction is a stable characteristic of a bacterium. Gram positivity (the ability to resist decolorization with ethanol or acetone) is a feature of relatively young bacterial cells of some species; as they age, the cells lose this characteristic and apparently become Gram-negative. It is important, therefore, to examine young cultures, preferably before the end of the logarithmic phase of growth. Genuinely Gram-negative bacteria do not retain the first stain which is easily removed by the decolorizing agent. Thus, as in many other tests, a positive finding (in this case retention of the purple stain) has much more significance than a negative result which may, in fact, be false due to (i) the age of the culture, or (ii) excessive decolorization with powerful solvents such as acetone. There are many variations of Gram's staining method (and each works well in the hands of those who practise it); the one we use under the name of Lillie's modification is simple and gives good results but, as acetone is used, the decolorization can be overdone. A modification by Preston & Morrell (1962) is claimed to be foolproof. Recently, a rapid paper-strip method has been 24

[4.3]

marketed for distinguishing between Gram-positive and Gram-negative organisms though it has doubtful practical and no taxonomic value. 4.4.2 Morphology is affected by the medium on which the organism is grown and by the temperature of incubation. Organisms are typical and in their most natural state in young cultures; in wet, unstained preparations, they are best observed by phase-contrast or dark-ground microscopy. Such examination will show not only the shape(s) of organisms but, when prepared from suitable material (see Section 3.3.5), will show motility if it is present, and whether the cells remain rigid (as in most bacteria), flex (spirochaetes), or glide (cytophagas). The distinction between spheres (cocci) and rods (bacilli) is not always clear-cut, and genera such as Acinetobacter and Moraxella cannot be placed categorically in one morphological group. Although it is usual to describe organisms of both these genera as either coccobacilli or short rods, electron micrographs clearly show the coccal nature of some acinetobacter strains (Thornley, 1967). Baumann, Doudoroff & Stanier (1968b) found that the differences in morphology of these genera corresponded with the growth phase: plump rods in the logarithmic phase and coccoid forms in the stationary phase. Brzin (1965) described what she called a sphaeroplasting effect, in that prolonged incubation of 'Acinetobacter anitratus' (Acinetobacter calcoaceticus) strains at 37 °C produced polymorphism. Bizarre-shaped cells may suggest particular genera and the presence of clubs or dumb-bell forms will call for staining by methods such as those of Neisser or Albert or with Loeffler's methylene blue, capable of showing metachromatic granules. Electron microscopy is still not yet available in all diagnostic laboratories and without it the site of insertion of bacterial flagella cannot be determined accurately. Fortunately such information is not often needed for the identification of motile bacteria, and we use it only for the genus Campylobacter for which the simple flagella stain of Kodaka et al. (1982) is recommended. Scanning electron microscopy seems to have special advantages in the identification of the actinomycetes (Williams & Davies, 1967).

PRIMARY TESTS USED IN THE TABLES

4.4.3 Acid fastness is shown when an organism resists decolorization by strong acids or mixtures of ethanol and mineral acid; this is characteristic of a few bacteria and when positive is diagnostic of mycobacteria. Nocardias are sometimes acid-fast but seldom resist the vigorous decolorization which mycobacteria successfully endure. The 'hot' staining method of Ziehl-Neelsen (Ziehl, 1882; Neelsen, 1883) is usually used to demonstrate acid fastness but 'cold' staining methods are also used (Aubert, 1950), as well as fluorescent techniques with auramine or rhodamine. 4.4.4 Spores are stained by a modification of Moeller's (1891) method (itself a modified ZiehlNeelsen method) in which ethanol is used for decolorization. The staining method is simple and seldom causes difficulty but young spores do not resist decolorization and they may or may not take up the counterstain. For clostridial spores, the malachite green method of Schaeffer & Fulton (1933) is preferred. In older cultures some bacilli may shed their spores so that in rod-shaped bacteria unstained areas occur and the stained spores may lie free of the cells from which they developed. As an alternative to staining, phasecontrast microscopy may be used. An indirect method of demonstrating the presence of spores is to show that a culture can survive heating at 80 °C for 10 minutes. A problem that faces the bacteriologist is the tendency for sporing organisms to lose the ability to produce spores. The asporogenous state may be permanent, or it may be a temporary reaction to a particular environment, when a change of medium or temperature of incubation could suffice to restore the strain's ability (or need) to form spores. Subculture to a starch-containing medium, such as Potato Agar, is often successful in restoring the ability of an aerobe to form spores; in other instances a deficiency of manganese in the medium may be the cause of the asporogenous state and the remedy is a supplement of 'trace elements'. Often the cause is unknown and the best general advice, based on the restoration of many asporogenous strains of Bacillus in the National Collection of Type Cultures to the sporing state, is to use a medium containing 'soil extract' (Appendix A2.6.35). The meaning of the term 'spore' in bacteriology has been the subject of much discussion. As a purist,

[4.4] R. E. Buchanan (see also Cross, 1970) insisted that bacterial spores were 'endospores' formed actively within the bacterial cell in contrast to the 'fragmentation spores' of Nocardia species. But most bacteriologists do not regard the nocardias as a sporing organism, and we think that the simple word spore tells us all we need to know about it. As we understand the bacterial spore, it is a body resistant to heat and disinfectants that is formed by relatively few bacteria; of the animal pathogens the only sporing bacteria are members of the genera Clostridium and Bacillus. Historically, anaerobic spore-forming bacteria were important in war wounds; their occasional involvement in a wide variety of clinical syndromes is, however, now recognized. Sterilization techniques are checked by including spore suspensions in drums containing dressings and gloves; some spores are more heat-labile than others, and spores of a resistant species (usually of Bacillus stearothermophilus) are used. After sterilizer tests, the spore suspensions returned to the laboratory should be incubated at about 50 °C and the cultures observed for at least a week. It is important to identify any strain that appears to survive sterilization; a mesophilic spore-former would suggest post-sterilization contamination. In connection with the acid-fast and spore-forming characters of organisms, certain other problems need to be discussed. Should every culture be stained by Ziehl-Neelsen's and Moeller's methods or tested for heat-resistance, or should these tests be restricted to Gram-positive organisms or to those cultures which, from their morphology, colony form, rate of growth, and other characters, we suspect may be acid fast or able to produce spores? We do not know of any Gram-negative bacteria that are genuinely acid fast and it is therefore reasonable to omit the ZiehlNeelsen stain for Gram-negative organisms. Should we stain all cultures for spores and, failing to find them, try again after further cultures on Soil Extract Agar or other spore-encouraging media? We know that these tests are not done as a routine and, as the majority of cultures will show negative results, we do not suggest that the search for sporing forms need be made in every case. All we would stress is that when spores are not looked for, a mental note should be made that they have not been excluded, and that their possible presence should be borne in mind. 25

BACTERIAL CHARACTERS AND CHARACTERIZATION 4.4.5 Motility may be studied in a hanging-drop or other wet preparation. Some strains are only sluggishly motile when first isolated; motility may be speeded by using Graigie's technique (Craigie, 1931; Tulloch, 1939) in which the organism is inoculated into a central tube of sloppy agar and, after incubation, a subculture is made from those organisms that, by their motility, have migrated outside the central tube. Motility may be inferred by observing the spreading growth in a semisolid agar (Tittsler & Sandholzer, 1936) which may be seen better when a tetrazolium dye is incorporated in the medium; as the organisms grow the dye is reduced, and the medium changes colour (Kelly & Fulton, 1953). The temperature of incubation is important; most motile organisms are motile at lower temperatures (e.g. 15-25 °C) and may not be motile at the temperature (e.g. 37 °C) optimal for growth. The problem pertaining to motility is: should all strains be tested or only rods? If we only examine the rods we shall overlook the motility of many strains of Enterococcus (Streptococcus) faecium, of Micrococcus agilis, and other cocci. When these tests become part of the daily routine they do not take up much extra time; they are only time-consuming and upsetting of routine when they are 'special tests'. These remarks refer to the motility shown by aerobic organisms; anaerobes present special problems in that motility will be inhibited by the air present in hanging-drop preparations. Capillary tube preparations, sealed at each end, from cooked meat cultures, are more likely to show motility in clostridia. Some bacteria (cytophagas) are motile by a gliding movement and special media and techniques are necessary to observe it. This type of movement is affected not only by the concentration of agar in the medium, but also by the concentration of peptone. Such organisms are not likely to be found in pathological specimens because the methods used by medical bacteriologists are not suitable for showing this gliding motility. Lautrop (1961), Halvorsen (1963) and Piechaud (1963) thought that they had found a similar motion in 'Bacterium anitratuni (Acinetobacter calcoaceticus) and Moraxella Iwoffii (Acinetobacter Iwoffii), but this was not true gliding motility and the organism should be regarded as non-motile (Lautrop, 1965). 26

[4.4]

4.4.6 The ability to grow in air is a character shared by all bacteria except anaerobes and strict microaerophiles such as campylobacters; it is a feature needed in Table 6.1 for the identification of certain anaerobes (especially Clostridium perfringens) in which spore formation may be difficult to show, and which, without this character, would appear to be placed among the lactobacilli, corynebacteria, or other Gram-positive rods. Failure to grow in air may be due to a deficiency of carbon dioxide, and growth in an atmosphere of air with added CO2 should be attempted. Three important clostridia, Cl. histolyticum, Cl. tertium and Cl. carnis, grow mod-erately well in air and are regarded as 'facultative aerobes'. All these difficulties can be largely overcome by relying on the universal sensitivity of anaerobes to metronidazole. Both aerobes and facultative anaerobes (whether CO2-dependent or not) are uniformly resistant to metronidazole under anaerobic conditions. 4.4.7 Ability to grow under anaerobic conditions is fairly widespread among bacteria but as it is not universal the knowledge that an organism cannot grow under these conditions can be diagnostically important. Some of these organisms are strict aerobes, others may need carbon dioxide for growth. In contrast the ability of some Bacillus species to grow anaerobically can also be diagnostically useful. 4.4.8 Carbon dioxide requirement. An incubator in which the concentration of CO2 can be regulated or an anaerobic jar from which the appropriate amount of air is evacuated and replaced with CO2 are necessary for defined conditions; if these are not essential, a candle jar can be used which gives an atmosphere of about 2.5% CO2 and 17% O2 (Morton, 1945); for anaerobes, a CO2 gas-generating kit in an anaerobic jar yields a final atmosphere of CO2 (10%) and hydrogen in the absence of oxygen. 4.4.9 The catalase test is simple and seldom causes difficulty, but because some strains of lactobacilli, pedicococci, and a few strains of Enterococcus (Streptococcus) faecalis appear to form catalase, Gutekunst, Delwiche & Seeley (1957) questioned the validity of the test 'as an overriding classification feature'. False catalase reactions by some lactobacilli grown in low (0.05%) concentrations of

PRIMARY TESTS USED IN THE TABLES

glucose (Dacre & Sharpe, 1956) are due to an azideinsensitive, non-haem catalase (pseudocatalase) and can be avoided by using media with 1% glucose without added haematin (Whittenbury, 1964). A few species (e.g. Aerococcus viridans) produce a weakly positive reaction which may easily be missed by those looking only for strong reactions. Gagnon, Hunting & Esselen (1959) described a simple method in which some of the growth of the organism under test was spread on discs of filter paper and dropped into 3% H2O2; when catalase was present the evolution of gas quickly brought the discs to the surface. Alternatively, a commercial paper-strip method is available for the detection and measurement of hydrogen peroxide production (Appendix C1.17). Those who work with mycobacteria have different criteria for the catalase test; the methods are semiquantitative and positive results are graded by the height of the column of bubbles in a standard test (see Appendix C3). Another method, the catalase drop test, can be used for rapid results. To reduce the danger from aerosols, as with cultures of Yersinia pestis, Burrows, Farrell & Gillett (1964) stab-inoculate agar containing 0.5% H2O2 and, using low power magnification, look for effervescence; if a disinfectant that lowers surface-tension (e.g. cetrimide, 0.5%) is added to the agar, the oxygen bubbles persist longer and further reduce the danger from aerosols. 4.4.10 The oxidase test was first used to demonstrate colonies of Neisseria species in mixed cultures (Gordon & McLeod, 1928; McLeod et aU 1934; McLeod, 1947), but following the test devised by Kovacs (1956) it is now used also to distinguish pseudomonads from the enteric bacteria. When precautions are taken to avoid oxidation of the reagent, the test is sensitive and useful in classification and identification (Steel, 1961). Leclerc & Beerens (1962) use a technique similar to Kovacs' but substitute the more stable dimethyl for the tetramethyl compound. Brisou et al. (1962) suggested a modification of Kovacs' method that is said to make the result of the test more clear-cut and easier to read. In the USA the term 'cytochrome oxidase' is used for the reaction, and the methods recommended are those of Gaby & Hadley (1957) and Ewing & Johnson (1960), which are less sensitive than Kovacs' method.

[4.4] Tests for oxidase resemble those for H2S in that methods differing in sensitivity can be used; as in the H2S test, greater bacterial differentiation can be obtained by using a method of low sensitivity. Steel (1962Z?) found Kovacs' method too sensitive for staphylococci, some of which gave weak or delayed reactions; he obtained taxonomically more helpful results by using Gaby & Hadley's (1957) method, with which all staphylococci were oxidase negative. However, with the description of many more species of staphylococci (e.g. S. sciuri), this is no longer the case. 4.4.11 The Oxidation-Fermentation (OF) test. To find out whether the attack on carbohydrates is by oxidation or by fermentation, the OF test is done by growing the bacterium in two tubes of Hugh & Leifson's (1953) medium; in one tube the medium is covered with a layer of soft paraffin (petrolatum). It is important to use a solid form as liquid paraffins cannot be expected to give comparable results. Oxidizers show acid production in the open tube only; fermenters produce acid in the paraffin-covered tube and, starting from the bottom, in the open tube. The usual sugar included in the Hugh & Leifson medium is glucose but, because of the occurrence of organisms which do not seem to attack glucose but break down other sugars (Hugh & Ryschenkow, 1961; Koontz & Faber, 1963) the need for a test using the basal medium with maltose or pentoses should be considered. Park (1967), who had some indefinite results in a modified Hugh & Leifson medium, developed a simple test to show that the glucose had been utilized and was no longer present in the medium. The OF basal medium may be used for all the 'sugar reactions' needed to characterize bacteria (Gilardi, 197 la). Various modifications to the medium have been suggested, including a useful peptonefree medium by Board & Holding (1960); to avoid confusion we do not use the term 'Hugh & Leifson test', but prefer the more descriptive term Oxidation-Fermentation (OF) test. With the OF test three points deserve mention, (i) The organism may not be able to grow in the Hugh & Leifson type medium, in which case the test must be repeated using a basal medium enriched with 2% serum or 0.1% yeast extract, (ii) The organism may 27

BACTERIAL CHARACTERS AND CHARACTERIZATION grow but not produce acid in either tube. This result should be confirmed by inoculation of Park's (1967) modified Hugh & Leifson medium containing glucose, (iii) Leifson (1963) found that bromothymol blue was toxic to some bacteria, and modified the OF medium for marine organisms. The OF test is one of the most important tests carried out in the early stages for identification of aerobic bacteria. Most genera are composed of bacteria that either oxidize or ferment glucose; when a genus contains some species that attack glucose by oxidation and other species by fermentation, there would seem to be good reason to reconsider the taxonomy of the genus and the desirability of dividing it. Some organisms do not appear to be able to attack a sugar readily, and often show acid production only after several days of incubation. Lederberg (1950) found that this delay was due to failure of the sugar to reach the inside of the bacterial cell, and the ONPG test (Section 4.5.35) was devised to reveal quickly the potential fermentative power of these 'late lactose fermenters'.

4.5

Secondary tests used in the tables

These notes give some background information about the secondary tests used. Details of special media used for the biochemical tests are given in Appendix A; the reagents and methods are described in Appendix C. 4.5.1 Acetylmethylcarbinol production. The Voges-Proskauer or VP test can be carried out in many ways and with almost any desired degree of sensitivity. It is now generally thought that the older methods (Harden & Norris, 1912) are too slow and insensitive, but there is less agreement about the method to be recommended or the sensitivity that gives the best differentiation between taxa. Although Clark & Lubs (1915) specified 30 °C for the test, the MR and VP tests were often carried out on cultures that had been incubated at 37 °C. For many years, water bacteriologists recommended 30 °C (Report, 1956&) and we now know that some enterobacteria such as Hafnia often give a negative VP result at 37 °C but a positive reaction at 30 °C or lower. In a comparative trial it was found that incubation for 5 28

[4.4]

days (at 30 °C) was the minimum time needed to detect by Barritt's method (1936) all the positive results among the enterobacteria (Cowan & Steel, 1964); for other organisms (e.g. staphylococci) longer incubation up to 10 days gave a greater number of positive results. Others have reported that acetylmethylcarbinol (acetoin) may be broken down and used as a carbon source by various coliform organisms (Linton, 1925; Paine, 1927; Ruchhoft et aL, 1931; Tittsler, 1938), Bacillus species (Williams & Morrow, 1928) and staphylococci (Segal, 1940). Taylor (1951) found that O'Meara's (1931) fumarate medium prevented the breakdown of acetylmethyl carbinol by soft-rot bacteria and allowed it to accumulate. Outside the field of enteric bacteria it has been found that phosphate may interfere with the production of acetoin; Smith, Gordon & Clark (1946) recommended a medium in which the phosphate is replaced by NaCl, and Abd-el-Malek & Gibson (1948&) used a simple glucose peptone broth without added salt or phosphate. Cowan & Steel (1964) compared Glucose Phosphate Broth with the media recommended by Smith, Gordon & Clark and by Abdel-Malek & Gibson, and they came to the conclusion that Glucose Peptone Broth was the most suitable medium for Bacillus and Staphylococcus and Glucose Phosphate Broth was best for the enterobacteria and most other organisms. After comparing different methods for the VP test for many years, Cowan & Steel (1964) chose Barritt's (1936) method as a satisfactory standard; the sensitivity was found to be mid-way between O'Meara's (1931) and Batty-Smith's (1941) methods. For a useful review of the VP test see Eddy (1961). It should be noted, however, that commercial kits usually employ pyruvate as the substrate for the VP test; this is more sensitive and yields more positive reactions than with Glucose Phosphate Broth, especially with streptococci. 4.5.2 Aesculin hydrolysis is a test of value for streptococci, many anaerobic genera and some other organisms. The glycoside aesculin contains molecules of the aglycone 6,7-dihydroxycoumarin and glucose; hydrolysis of aesculin may be demonstrated in one of two ways. The usual method is to incorporate the glycoside in a nutrient base together

SECONDARY TESTS USED IN THE TABLES

with a ferric salt; aesculin hydrolysis is indicated by a brown coloration due to reaction of the released aglycone molecule with the iron. In addition, hydrolysis of aesculin, which is naturally fluorescent in UV light, can be confirmed by the loss of fluorescence, thus obviating possible confusion with pigmentproducing organisms. Alternatively, utilization of the related glucose portion of the aesculin molecule by the organism can be detected by acid or acid and gas production. 4.5.3 Bile solubility is used to distinguish pneumococci from the viridans types of streptococci; however, the test is not specific for Streptococcus pneumoniae. The pneumococcus differs from other streptococci in having an autolytic enzyme which can be demonstrated by allowing a digest broth culture to age in the incubator; at 24 hours the broth is turbid but after a few days the medium becomes clear owing to lysis of the bacterial cells. Bile and bile salts activate the autolytic enzyme, and thus speed up cell lysis. They will not, however, produce clearing of a heat-killed culture or one that is too acid; the suspension to be tested should therefore be about pH 7.2. At one time crude bile was used for the test but the isolation of various bile salts in a pure state showed that certain of them were more active than others (Downie, Stent & White, 1931). Sodium deoxycholate is satisfactory and can be obtained in a reasonable state of purity. 4.5.4 Buffered single substrate (BSS) tests are, as their name implies, attempts to avoid the usual biochemical tests made in complex media with the concomitant risk that metabolic products may interfere with the specific reactions under test. The risk is real and is most obvious in sugar reactions tested in peptone-containing media on organisms that oxidize carbohydrates; for example, as long ago as 1955 Pickett showed that brucellas were able to produce acid from several sugars in an otherwise inert milieu. Various methods have been used to exploit the BSS principle; tablets containing substrate and buffer (Hoyt, 1951; Pickett & Scott, 1955; Hoyt & Pickett, 1957) were dissolved in small volumes of water, steamed, and the solutions heavily inoculated with the test organism. As the biochemical tests were carried out within a few hours, the results probably

[4.5] depended on the presence of preformed enzymes. The micromethods of Clarke & Cowan (1952), which largely depended on preformed enzymes, had the disadvantage that the preparation of the heavy suspensions of test organisms involved too many manipulations for use in a routine laboratory. Pickett (1970) and Pickett & Pedersen (19706) have developed a series of tests (or rather of test surroundings) in which the organism can act on one substrate at a time; the principle is applied to sugar reactions, decarboxylases, deamidases, and hydrolases. The tests are particularly useful for characterizing non-saccharolytic bacteria and those that oxidize carbohydrates - the so-called non-fermenters. We should point out that characterizations based on BSS tests will not always be the same as those made by conventional methods, nor will they match the characters shown in most of our tables (cf. Tables 1.6a,b). 4.5.5 A positive CAMP test, described by Christie, Atkins & Munch-Petersen (1944), is the production of a clear zone around a colony in an area of a blood agar plate that has been affected by staphylococcal (3-toxin; this bald statement needs amplification, for the clearing takes place only on blood agar made with sheep or ox blood, and not on media made with human, rabbit, horse, or guinea-pig blood. The important point in carrying out this test is that the agent produced by the bacterial cells must come in contact with the sheep (or ox) red cells before the staphylococcal (3-haemolysin. The test is almost specific for strains of Streptococcus agalactiae from man or animals; Christie, Atkins & MunchPetersen (1944) failed to find any other streptococcal species that produced the clear zone, but some haemolytic strains of groups E, P, and U give positive CAMP reactions (Shuman et al.9 1972). Pasteurella haemolytica also gives a positive reaction (Bouley, 1965). A modified CAMP test utilizing a culture of p-haemolytic group B streptococci on horse Blood Agar is of value in the identification of strains of Cl. perfringens which produce oc-toxin (Gubash, 1978, 1980). Fraser (1961) described a somewhat similar synergistic haemolytic effect that may occur when Corynebacterium pseudotuberculosis and Rhodococcus

equi are grown together on Blood Agar made with washed blood cells from sheep but not from 29

BACTERIAL CHARACTERS AND CHARACTERIZATION the horse. Unlike the CAMP phenomenon this observation does not seem to have led to the development of a useful specific diagnostic test. 4.5.6 Carbohydrate breakdown. The division of bacteria into fermenters, oxidizers, and non-utilizers by the OF test of Hugh &Leifson (1953) is one of the most heavily weighted of the primary tests used in the progressive system of identification in this Manual, and carbohydrate utilization also features in the secondary tests. The latter so-called 'fermentation tests' were used by early bacteriologists to distinguish one organism from another and elaborate diagnostic tables were based on them (see, for example, Castellani & Chalmers, 1919). The introduction of the simple inverted inner tube for gas collection (Durham, 1898) and the use of pH indicators enabled the production of gas and acid to be detected by inspection. Screw-capped bottles and tubes are not satisfactory for sugar tests because the CO2 evolved by the bacteria during growth is trapped and, by lowering the pH value of the medium, may change the colour of the indicator and suggest a (false) positive result. If screw-capped containers are used, the caps should therefore be loosened about an hour before the indicator colour is observed. Too much emphasis is possibly placed on bacterial differentiation by the reactions of individual sugars (apart from glucose; Section 4.4.11). In classification by numerical methods the composition of taxa is essentially the same whether or not the sugar reactions are included in the characters analysed (Focht & Lockhart, 1965). In the genus Acinetobacter acid production from several sugars is mediated by a nonspecific aldolase (Baumann, Doudoroff & Stanier, 19686). The failure to standardize methods has led to discrepant results in the hands of different workers, and it is only within recent years that taxonomists have given adequate thought to the significance of acid production by a bacterium growing in a medium containing a carbohydrate. Peptones are also present in such a medium and, during growth of the organism, these are broken down to substances that are alkaline in reaction; if, in the medium, there is a carbohydrate, alcohol, or other substance commonly called a 'sugar' that can be broken down by the bacteria either by oxidation or by fermentation, acid will be pro30

[4.5]

duced, but it will be detected by a pH indicator in the medium only when the acid produced from the sugar exceeds the alkali from the peptone. The visibility of the reactions is also influenced by (i) the buffering properties of the medium, and (ii) the indicator used; for example, bromthymol blue shows acid production when the pH value falls to 6.0 or less, whereas bromcresol purple does not change colour until the pH has fallen to about 5. Peptone Water Sugars, which are commonly used in the UK, have less buffering power and yield less alkali than the broth-based sugars used extensively in the USA and elsewhere. With Peptone Water Sugars we prefer Andrade's indicator (which becomes pink at about pH 5.5) or bromcresol purple (yellow at about pH 5), and it is fortunate that in these media the enterobacteria give similar results to those with broth-based sugars containing bromthymol blue (yellow at about pH 6.0). With apparently non-saccharolytic bacteria the results obtained in peptone-containing sugar media may be misleading in assessing their carbohydrateattacking ability because a small amount of acid produced will be masked by the breakdown products from peptone. Pickett (1955) and Pickett & Nelson (1955) overcame this difficulty by using buffered peptone-free media; with cresol red as indicator they showed that acid was produced from several sugars by the apparently 'non-fermenting' Brucella species. The development of these methods (Pickett, 1970) is outlined in Section 4.5.4. Another method to reveal the acid-producing potential of 'Acinetobacter anitratus' (Acinetobacter calcoaceticus) (Stuart, Formal & McGann, 1949) and of slow lactose-fermenting coliform organisms (Lowe & Evans, 1957) is to increase the carbohydrate concentration to five or even ten per cent. A peptone-free modification of the Hugh & Leifson (1953) OF medium has been suggested (Board & Holding, 1960; Holding & Collee, 1971) in which acid production correlates well with utilization of glucose; it can be used for aerobic, oxidizing bacteria, and also for bacteria that require additional growth factors. Some bacteria will not grow on simple media and need an enriched sugar medium. Many streptococci and corynebacteria are grown in media containing serum; neisserias in media enriched by serum or ascitic fluid; and haemophilic bacteria in sugar media to which X and V factors have been added.

SECONDARY TESTS USED IN THE TABLES

Organisms that oxidize sugars do not readily show acid production when they are grown in tubes of liquid media, and more reliable results are obtained when they are grown on the surface of solid media, thus exposing the organisms to an adequate supply of air. Oxidizers such as Pseudomonas species do not give reliable 'sugar reactions' on peptone-containing media and they should be grown on media with an ammonium salt as the main nitrogen source. Smith, Gordon & Clark (1952) used ammonium salt sugars (ASS) for their work on Bacillus species, some of which ferment and others oxidize carbohydrates. Bacteria may be grown on peptone-containing broth and then centrifuged and resuspended in water or saline so that, when added to a buffered solution of carbohydrate, the bacterial enzymes act on a single substrate (see, for example, Davis, 1939; Clarke & Cowan, 1952; Le Minor & Ben Hamida, 1962). Snell & Lapage (1971) compared four methods of detecting glucose breakdown, one of which was positive only when all the glucose had been utilized. Peptone water sugars gave the fewest positive results and the ammonium salt sugars the most; a modified OF medium (Park, 1967), tested for residual glucose, gave results almost as good as those with ASS. Snell & Lapage (1971) confirmed that Pseudomonas maltophilia could attack glucose when methionine, an essential growth factor, was added to the ASS medium, thus showing that it was suitable, when supplemented, for testing fastidious bacteria. Carbohydrate fermentation by anaerobes can be demonstrated in liquid media incubated under anaerobic conditions although gas production alone is unreliable as an indicator since it is often evolved also from proteins. As anaerobiosis decolorizes pH indicators, it is usual to add them after incubation. Many anaerobes do not grow satisfactorily in ordinary liquid sugar media, and it is necessary to use an enriched basal medium for fermentation tests. The horse blood agar plate-fermentation method of Phillips (1976) is consistently successful, giving rapid and unambiguous positive or negative results. 4.5.7 Carbon source utilization (CSU) tests are used extensively in classification work; their application to identification is limited mainly to tests for the utilization of citrate. The mineral basal medium used by Owens & Keddie (1969) for studies on nitrogen

[4.5] nutrition can be used for the CSU tests; it may be supplemented when necessary with an amino acid mixture or with yeast extract (0.02%) and vitamin B 12 (2u£/Htre). Gordon & Mihm (1957) used organic acids as carbon sources in the basal medium described in Section A2.6.8. But relatively few CSU tests are used in routine diagnostic laboratories, although commercial kits are available for non-fermenters and yeasts. Citrate utilization is tested in Koser's (1923) citrate medium or in a similar medium solidified by agar (Simmons, 1926). Vaughn et al. (1950) believed that the addition of agar invalidated the test. The medium must be in chemically clean tubes (see Appendix A 1.1). In tests of this kind the inoculum should be small and free from medium on which the organism has grown. To avoid carry-over, use a straight wire instead of a loop, and inoculate from a dilute suspension in water, saline or buffer. All growth should be confirmed by subculture (again using a straight wire) to another tube of the same citrate medium. Other citrate media, such as Christensen's, contain additional nutrients and do not test the ability of the organism to use the citrate radical as the sole carbon source. An organism growing in Koser's or Simmons' medium will grow on Christensen's medium, but one growing on Christensen's medium may not grow on the other two media (but see Piechaud & Szturm-Rubinsten, 1963). 4.5.8 Chitinolytic activity can be shown as a clear halo around growth on Chitin Agar. As chitin is insoluble in water a purified preparation is suspended in Salt (for halophiles) or Water Agar and layered on a base of Nutrient Agar (Appendix C3.11). 4.5.9 The coagulase test was developed from observations that certain staphylococci clotted plasma from the goose (Loeb, 1903), man, horse, and sheep (Much, 1908); Gratia (1920) introduced the name staphylocoagulase for the active agent. At least two substances, bound and free coagulases, are concerned, but the tube methods for carrying out the coagulase test do not distinguish between them; the slide test (Cadness-Graves et al., 1943) detects bound coagulase. A growing number of commercial preparations are available for testing coagulase production, including both bound and free forms. 31

BACTERIAL CHARACTERS AND CHARACTERIZATION The type of plasma used in the test may affect the result; the anticoagulant should not be citrate alone for this will be removed by citrate-using bacteria such as 'KlebsieUa aerogenes (K. pneumoniae subsp. aerogenes) (Harper & Conway, 1948) or certain streptococci (Evans, Buettner & Niven, 1952), with the result that a clot will form after prolonged incubation and give the (false) appearance of a delayed positive coagulase test. Harper & Conway (1948) recommended that heparin should be added to citrated plasma to prevent clotting of fibrin when the citrate is withdrawn. A filterable coagulase-like factor produced by some streptococci will not clot heparinized plasma (Wood, 1959). The species of animal from which the plasma is derived is important: for staphylococci of human origin, plasma from man or rabbit should be used; sheep and bovine plasma give fewer positive results and guinea-pig plasma gives even fewer. When strains from animals other than man are under test, it is advisable to use plasmas from several animal species, including the one from which the strains were isolated. The coagulase test is simple, so simple that there are almost too many ways of doing it. Williams & Harper (1946) compared many of these methods, including that recommended internationally for testing for free-coagulase (Subcommittee, 1965), and those given in Appendix C 3.12 are based on their conclusions as well as our own experience. We draw attention however to a few important points. Occasionally a strain will be isolated that produces so much fibrinolysin early in its growth that a clot from coagulase action never becomes visible. Sometimes a small clot forms early but lyses quickly; for this reason a reading should be made an hour after starting the test. Some strains produce only small amounts of coagulase and the clot may only be seen after overnight incubation. Each batch of plasma should be tested to confirm that it is suitable for the coagulase test; filtered plasma is generally unsuitable. Positive and negative controls should be included in the tests put up each day. The use of fibrinogen was investigated by Cadness-Graves et al. (1943) and was found to be a suitable substitute for plasma. Reconstituted dried plasma can also be used (Colbeck & Proom, 1944). 4.5.10 Decarboxylases for amino acids are characteristic for different bacterial groups among the 32

[4.5]

Enterobacteriaceae (M0ller, 1954a, c). Initially the determination of the decarboxylases was a research problem but M0ller (1955) developed simpler technical methods so that the decarboxylase pattern became a useful taxonomic tool at a higher level than antigenic structure. The method is not specific for decarboxylation and may indicate other metabolic processes such as deamination, deamidation, and transamination (Cheeseman & Fuller, 1966); however, these distinctions are not made in this Manual. Glutamic acid decarboxylase, which is characteristic of Escherichia, Shigella, Providencia, Morganella and Proteus, cannot yet be determined simply, but arginine dihydrolase, and lysine and ornithine decarboxylases can now be detected readily by observing the colour change of an indicator. Falkow (1958) introduced even simpler tests but they were not satisfactory with klebsiellas; they cannot therefore be used with organisms of unknown identity. Arginine is hydrolysed by some but not all streptococci and corynebacteria (Niven, Smiley & Sherman, 1942); methods of detecting arginine hydrolysis by pseudomonads are described by Sherris et al. (1959) and Thornley (1960). Steel & Midgley (1962) found the decarboxylase patterns of different genera and species taxonomically useful. M0ller's methods are not always satisfactory with the non-fermentative bacteria, and BSS decarboxylase tests (Pickett & Pedersen, 1970&) may give a greater number of positive results. 4.5.11 Denitrification. Many bacteria isolated in medical and veterinary laboratories are denitrifiers, that is, they not only reduce nitrate to nitrite but also reduce nitrite to nitrogen gas. This in itself upsets the reading of the nitrate reduction test (see Section 4.5.31 and Table 4.1). The gas may be detected in an inverted inner (Durham) tube in Nitrate Broth or in the butt of slopes of the Fluorescence-denitrification (FN) agar medium of Pickett & Pedersen (1968). 4.5.12 Deoxyribonuclease (DNase) can be shown by growing the test organisms as streaks on or as stab-inocula in a medium (either defined or complex) containing DNA, and after incubation for 36 hours, flooding the plate with 1 N-HC1. A clear zone around the growth indicates DNase production. The temperature of incubation may be important. Jeffries, Holtman & Guse (1957) advise incubating at several

SECONDARY TESTS USED IN THE TABLES

temperatures as the enzyme may act at a temperature other than that for optimum growth; with Bacillus megaterium they obtained zone sizes of 0.2 cm at 37 °C, 0.5 cm at 30 °C, and 0.9 cm at 25 °C. Alternatively, decolorization of toluidine blue or methyl green by the breakdown products of DNA is a sensitive and suitable method for organisms such as campylobacters and staphylococci (Gilardi, 1978). The presence of DNase is often used in clinical laboratories for the presumptive characterization of Staphylococcus aureus, either as well as or instead of coagulase for presumptive pathogenicity. 4.5.13 Digestion of meat, inspissated serum, Dorset Egg medium or casein are used as indicators of proteolytic activity. At one time gelatin liquefaction was used to detect proteolysis, but gelatinase is not a true proteolytic enzyme. 4.5.14 Ethylhydrocuprein or optochin inhibition of pneumococcal growth was described by Moore in 1915, but as a diagnostic test it has had a chequered career. Soon after its introduction it fell into disrepute and, as a means of distinguishing pneumococci from viridans streptococci, it was superseded by the bile solubility test, especially when the more highly purified bile salts became available (Downie, Stent & White, 1931). The optochin test has since come into its own again in the form of absorbent paper discs impregnated with ethylhydrocuprein for direct application to inoculated plates (Bowers & Jeffries, 1955; Bowen et al., 1957). In the concentration recommended by Bowers & Jeffries for direct application to inoculated plates, a small zone (1-2 mm beyond the disc) of inhibition may occur with a few viridans streptococci but pneumococci are inhibited more obviously with zones extending to 5 mm or more. The advantage of the optochin test over bile solubility is that the disc can be applied to any plate culture of the organism under test, whereas for bile solubility the suspension or broth culture must be of about neutral pH value. If small zones of inhibition are ignored and 5 mm is the minimum zone recorded as positive, the specificity of the optochin test for the pneumococcus is high. Bowers & Jeffries (1955) found that only one of 243 pneumococci failed to be inhibited; the exception was found to be avirulent for mice and was thought to be in the R form (though R pneumococci are bile-soluble).

[4.5] 4.5.15 Biochemical tests in enriched media. Delicate or fastidious organisms are often grown in media enriched with serum or other supplements which can introduce uncontrolled and uncontrollable side-effects. Whenever such supplements are used the enriched medium (without the test substrate) should be used as a control. For example, Cobb (1966) reported that Corynebacterium bovis produced ammonia from Christensen's urea medium supplemented with 3% serum. In a control experiment he found that the organism produced acid from the basal medium without urea (the basal medium contained glucose); when urea was present the test culture produced an alkaline reaction, confirming that alkalinity (the positive urease indicator) of the test was due to urea breakdown. An alternative method of dealing with fastidious organisms is to culture them on a suitable medium, wash off the growth with water or saline, centrifuge it, resuspend in water and use the suspension in a BSS test (Section 4.5.4). In this way Haemophilus influenzae has been shown to produce a powerful urease (Sneath & Johnson, 1973). Similarly, Helicobacter pylori was described as urease-negative until Langenberg et al. (1984) showed that it gives a rapid reaction in a BSS test with Christensen's urea broth. 4.5.16 Gelatin hydrolysis or liquefaction is shown by a test in which the organism grows in a nutrient medium solidified by gelatin; the disadvantages of the liquefaction test are that: (i) different samples of gelatin vary in gelling power; (ii) the cultures are incubated at a temperature (22 °C) below the melting point of the medium (about 25 °C) so that mesophilic organisms may grow very slowly or not at all; (iii) some bacteria will not grow in the medium. To overcome the second of these difficulties the cultures may be incubated at the optimal temperature for growth and later refrigerated to see whether the gelatin has retained its gelling property; uninoculated medium incubated in parallel acts as a control. Gelatin stab cultures may need weeks of incubation before showing liquefaction. Hucker (1924a), working with micrococci, found a curious relationship between the length of incubation and the first appearance of liquefaction; when the number of liquefying strains was plotted against duration of incu33

BACTERIAL CHARACTERS AND CHARACTERIZATION bation there were two peaks, one after about 1-2 weeks, and the second after about 3 months. The significance of tests of such long duration is doubtful (the gelatin may be denatured) and they are quite useless in identification work. However, the gelatin stab test should not be discarded; some species will liquefy it overnight, others will take longer, and these differences may be helpful in distinguishing between species such as Enterobacter cloacae, which takes a week or more, and Serratia marcescens, which liquefies gelatin in 1-2 days. For identification work the duration of incubation must be limited to a reasonable period, and one of the rapid methods should be used in parallel. Frazier's (1926) test has the advantage that the gelatin is in agar and the medium does not melt at 37 °C. After growth of the organism, the plate is flooded with an acid mercuric chloride solution which reacts with the gelatin in the medium to produce an opacity; where gelatin has been hydrolysed the medium remains clear. Frazier's (1926) method is preferred for anaerobes though it is now considered safer to use 30% trichloracetic acid instead of the acid solution of mercuric chloride. A rapid method devised by Kohn (1953) uses gelatin—charcoal discs hardened by formaldehyde; these do not melt at 37 °C and can be added to Peptone Water cultures, which are then returned to the incubator; preformed or induced enzyme will hydrolyse the gelatin and liberate the charcoal particles. Lautrop (1956a) found that the action of gelatinases was influenced by the presence of calcium ions and recommended that test organisms should be suspended in physiological saline with 00.1 M-CaCl2. Greene & Larks (1955) devised an even quicker micromethod in which Kohn's discs were used (Appendix C3.23). Thirst (1957Z?) and Hoyt & Pickett (1957) developed microscope-slide techniques which were similar to one described by Pickford & Dorris (1934), who found that the gelatin of photographic plates and film could be removed by proteolytic enzymes and bacteria. LeMinor & Piechaud (1963) describe a method in which the silver sulphide of exposed and developed film can be seen to be released when the gelatin is liquefied. 4.5.17 Gluconate is converted by some bacteria to 2-ketogluconate, which can be detected by the appearance of a reducing substance in the medium. 34

[4.5]

Haynes (1951) found this test helpful in identifying Pseudomonas aeruginosa. Shaw & Clarke (1955) simplified the test and reported that it was useful for klebsiellas. When Klebsiella species were compared with Enterobacter, Cowan et al. (1960) found that klebsiellas with IMViC reactions - - + + and Enterobacter species were gluconate-positive, but klebsiellas with other IMViC reactions were often gluconate-negative. 4.5.18 Growth or failure to grow on specified media can indicate (i) nutritional needs; (ii) sensitivity or insensitivity to substance(s) in the medium; and (iii) ability to use a specified compound as source of a particular nutritional factor. Examples of the characters revealed are: (i) growth on Blood Agar but not on the basal medium indicates a need for enrichment with blood; (ii) (a) failure to grow on MacConkey Agar, accompanied by growth on Nutrient Agar, shows sensitivity to bile salts; (b) growth on media containing 6.5% NaCl shows an unusual degree of salt tolerance; (iii) failure to grow on Koser's Citrate medium shows that the organism cannot use citrate as a carbon source under the test conditions. These utilization tests must be adequately controlled to prevent carry-over from the medium on which the inoculum was grown (see Citrate utilization, Section 4.5.7). 4.5.19 Haemolysin production and haemolysis are not always cause and effect; the ability to produce a soluble haemolysin is not necessarily associated with zones of haemolysis on Blood Agar plates (Elek & Levy, 1954). Streptococci produce haemolytic zones on the surface of Blood Agar made from the blood of most animal species and these organisms are rightly named haemolytic streptococci. The haemolysins produced by streptococci may be oxygenlabile (streptolysin O) or oxygen-stable (streptolysin S) and they need different conditions for their production; on Blood Agar plates, however, they produce similar zones of haemolysis. Streptolysin O is an antigenic extracellular protein; streptolysin S is non-antigenic, cell-bound and requires for its release a 'carrier' agent contained in serum or starch. Brown (1919) studied the nature of the haemolytic zones around streptococcus colonies in poured plates and labelled the types of haemolysis a (green zone, cell envelopes intact), (3 (clear, colourless zone, cell

SECONDARY TESTS USED IN THE TABLES

envelopes disrupted) and y (no action on red cells). The term y-haemolysis is an anachronism for 'nonhaemolytic' and describes a negative result. The application of the terms a and p has been extended to the haemolytic zones seen around bacterial colonies on the surface of Blood Agar, and though not in strict accord with Brown's usage, it is a convention that is well understood and is included in Table 63b. The P-haemolysis seen on Blood Agar plates is usually due to streptolysin S: some strains of S. pyogenes produce only the O haemolysin and are consequently non-haemolytic on Blood Agar unless incubated anaerobically. Streptococci that produce ochaemolysis or green zones on Blood Agar are often described as 'greening' or 'viridans' streptococci. The species name 'Streptococcus viridans,' previously attached to several different kinds of greening streptococci never adequately characterized, is not now used. Indeed Sherman (1937) applied the epithet usefully to describe the 'viridans' group of streptococci which are commensal rather than pathogenic. Streptococcus mitior is probably most representative of the organisms previously named *S. viridans'. Many of the viridans streptococci are unable to break down the hydrogen peroxide which they produce and this may contribute to the 'greening' phenomenon on Blood Agar medium as well as interfering with haemolysin activity and affecting their viability. Staphylococci behave differently on plates made with the blood of different animal species and it is misleading to speak of haemolytic staphylococci because the haemolysis may be due to a haemolysin or to a lipolytic enzyme (Orcutt & Howe, 1922). The soluble haemolysins can be used to detect the toxins produced by some strains of staphylococci; thus rabbit cell haemolysin is one manifestation of a-toxin, and sheep cell 'hot-cold' lysin is a characteristic of p-toxin. These toxins are not used in characterizing different species of Staphylococcus and so do not appear in the diagnostic tables. As with staphylococci, the haemolytic activity of the different haemolysins produced by strains of Clostridium depends partly on the species of red cells used. Thus, the a-toxin of CL perfringens, which is a 'hot-cold' lysin, is relatively inactive against horse erythrocytes, but is highly lytic for sheep red cells. The 6-toxin (oxygen-labile haemolysin) of CL perfringens, however, is strongly haemolytic for the ery-

[4.5] throcytes of both these animals. Because some of the clostridial haemolysins are species-specific, these toxins could be used to show the finer subdivision of the genus. Synergistic haemolysis on horse blood agar by ot-toxin-producing CL perfringens and phaemolytic group B streptococci forms the basis of the so-called CAMP test for the confirmation of CL perfringens (Gubash, 1980). The haemolytic activity of certain vibrios has some distinguishing value when tested with special media containing red blood cells from particular animal species (Sakazaki, Tamura & Murase, 1971; Barrett & Blake, 1981). 4.5.20 Hippurate may be hydrolysed to benzoate by bacterial action, and the ability to do this is limited to certain bacteria, for which it is an important characteristic. The end product is tested for by the addition of ferric chloride, which precipitates both hippurate and benzoate but the hippurate is more readily soluble in excess. The final concentration of iron is critical; to find the optimal amount of FeCl3 required, uninoculated tubes of medium, on which a titration can be made, should be incubated with the test cultures. The methods of Ayers & Rupp (1922) and Hare & Colebrook (1934) for streptococci use a relatively rich basal medium in which the organism will grow without hippurate. The Hajna & Damon (1934) method for coliform organisms uses Koser's medium (without citrate) as a base and so becomes a test of the organism's ability to use hippurate as a source of carbon as well as its ability to hydrolyse it. Thirst (1957a) added an indicator so that the growth may be seen more readily. The rapid test of Hwang & Ederer (1975) was originally described for group B streptococci, but it is a convenient method for campylobacters. 4.5.21 Hydrogen sulphide production by bacteria is such a common feature that, of itself, it has little differential value. The H2S test is one that can be made as sensitive as required (for a review see Clarke, 1953a); with an adequate sulphur source (cysteine) and a delicate indicator (lead acetate papers) almost all the enteric bacteria can be shown to be able to produce H2S. Tested in this way an accurate estimate can be obtained of an organism's catabolic power in relation to sulphur compounds, 35

BACTERIAL CHARACTERS AND CHARACTERIZATION but it is not possible to distinguish readily between those organisms with much and those with little ability to produce H2S. With a poor medium or a less sensitive indicator (ferrous chloride or lead acetate in the medium) only the strong H2S-producers are detected. This kind of test of low sensitivity allows clear distinctions to be made between Escherichia and Salmonella and even between different salmonellas. Two media yield results of this kind: Ferrous Chloride Gelatin and Triple Sugar Iron Agar (TSI). Both are recommended by the international Enterobacteriaceae Sub-Committee (Report, 1958) and their formulae are given in Appendix A, Lead acetate papers are not only ten times more sensitive than lead acetate in the medium, but they eliminate the toxicity of lead for the growing bacteria (ZoBell & Feltham, 1934). In the Brucella group the time of H2S production may be significant; this is found by changing the lead acetate papers each day. 4.5.22 Indole is volatile and can be detected either by testing the medium with /?-dimethylaminobenzaldehyde or by a paper strip impregnated with oxalic acid held near the mouth of the container by the cap or stopper. Both methods are sensitive and usually give the same result; occasionally all the indole volatilizes and only the paper strip is positive. Extraction of the indole from the liquid culture increases the sensitivity of the test; ether, xylol and petroleum have been used, but all are potentially dangerous if, following the usual bacteriological techniques, the mouth of the tube is flamed. Kovacs' (1928) reagent has the advantage that the solvent (amyl alcohol) is present in the test solution. Oxalic acid papers (Gnezda, 1899; Holman & Gonzales, 1923) and papers soaked in /7-dimethylaminobenzaldehyde (Kohn, 1954) are both sensitive indicators for indole. Temperature of incubation may affect the result; Taylor (1945-6) found three strains of Escherichia coli that were indole-negative at 37 °Cbut positive at 30 °C. Some organisms (e.g. Clostridium species) may break down indole; Reed (1942) found that with some species this happened so slowly that indole could always be detected in cultures 1-10 days old, but Clostridium sporogenes used it so quickly that it gave negative results when cultures were grown for only one day in a medium containing 1 mg indole per 100 ml broth. 36

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Unless indole has previously been extracted with xylol, sodium nitrite present in broth may interfere with its detection by Ehrlich's or Kovacs' reagents (Smith, Rogers & Bettge, 1972). 4.5.23 Inhibition of bacterial growth by a defined or a biological agent may be a useful identifying characteristic and is used in several of the tables; it occurs in different forms, ranging from inhibition by chemicals such as KCN or dyes to inhibition by bile salts or antibiotics. Inhibition by KCN (Appendix C3.32), optochin (C3.22) and the pteridine derivative 0/129 (C3.41) are described in the sections indicated. Commercially available antibiotic discs keep pace with prevailing fashion; they are available as single discs containing different amounts of antibiotic and as 'rings' with multiple discs for several antibiotics or different concentrations of them. Antibiotic sensitivity results are clearly important clinically but have only limited potential in helping to identify the organism isolated (see Section 4.3.1). 4.5.24 The KCN test distinguishes those bacteria that can grow in the presence of cyanide and those that cannot grow in the stated concentration. When a strain is reported as KCN positive it means that it grows in M0ller's (1954Z?) KCN medium and that it is therefore resistant to the concentration used. KCNnegative strains, i.e. those that do not grow, should be subcultured to the basal medium without KCN; if they cannot grow in the basal medium the test is without significance. M0ller (1954Z?) used waxed corks to prevent loss of cyanide from the tubes; these are unpleasant to handle and the use of small screwcapped bottles is preferable (Rogers & Taylor, 1961). Those who use the test assume certain responsibilities for safety; KCN and HCN are extremely toxic and the KCN solution should be kept in a locked cupboard. After use, the cyanide in the medium should be destroyed by adding ferrous sulphate and alkali before the tubes or bottles are autoclaved. 4.5.25 Lecitho-vitellin (LV) is the lipoprotein component of egg-yolk and it can be obtained as a clear yellow liquid by mixing egg-yolk with saline (Macfarlane, Oakley & Anderson, 1941). This liquid becomes opalescent when mixed with certain bacterial toxins or lecithinases; flocculation and separation

SECONDARY TESTS USED IN THE TABLES

of a thick curd of fat may follow. When lecithinaseforming organisms grow on a solid medium containing LV, the lecithinase diffuses into the agar and produces zones of opalescence around individual colonies. This reaction can be inhibited by adding certain antitoxic or antilecithinase sera to the surface of the medium before inoculation. Lipolytic organisms also produce an opalescence on LV Agar and it is often accompanied by a distinctive 'pearly layer' or iridescent film; the presence of free fatty acid can be demonstrated by treating the medium, after incubation, with copper sulphate solution (Willis, 1960). The ability to produce an opacity on LV Agar is useful in the division of the genera Bacillus and Clostridium, but other organisms, such as Staphylococcus aureus, may also give positive reactions. Egg-yolk emulsified in an equal volume of sterile saline is a practical and satisfactory substitute for LV for incorporation in solid media such as EggYolk Agar (McClung & Toabe, 1947). The LV reaction is not due solely to a lecithinase; Willis & Gowland (1962) consider that separation of insoluble protein, splitting of fats from lipoprotein complexes, and the coalescence of particles of free fat are all involved. In many laboratories media containing human serum (Nagler, 1939) have been replaced by egg-yolk media. Although Pseudomonas aeruginosa is known to produce a lecithinase, the egg-yolk reaction is usually negative; from this Stanier, Palleroni & Doudoroff (1966) argued that the egg-yolk reaction is specific for only one kind of lecithinase and that other types do not produce opacity with egg-yolk. The actions of four types of lecithinase are discussed by Willis (1969); only one (lecithinase C) is usually produced by bacteria. 4.5.26 The malonate test was introduced by Leifson (1933) to help distinguish Escherichia coli from 'Klebsiella aerogenes' (K. pneumoniae subsp. aerogenes), and with these organisms he found a perfect correlation with the VP test. Shaw (1956) showed that most strains of the Arizona group were malonate positive and most other kinds of salmonella were malonate negative. As both these groups are VP-negative there is thus no correlation between the malonate and VP tests. The test was described as a fermentation by Leifson and as a utilization by Shaw

[4.5] in spite of the fact that she added yeast extract to stimulate growth. 4.5.27 Metabolic fatty acids. The metabolic products that have received most attention, especially among the anaerobic bacteria, are the volatile fatty acids of the series formic to heptanoic, certain other short-chain carboxylic acids (notably lactic and succinic acids) and the low-molecular-weight alcohols. Minute quantities of these compounds may be analysed by gas-liquid chromatography (Holdeman, Cato & Moore, 1977). Qualitative and quantitative differences in end-products of metabolism are associated with different genera and species, and since these characteristics are stable they are of considerable taxonomic value for certain organisms (e.g. Fusobacterium). 4.5.28 The methyl red (MR) test, and the Voges-Proskauer (VP) test for acetylmethylcarbinol or acetoin, may be carried out with the same liquid culture. The tests are mainly used to distinguish various coliform organisms from each other; all these ferment glucose vigorously and the pH value of the glucose medium falls quickly. When methyl red is added after overnight incubation the cultures of all these organisms will give an acid reaction with the dye, i.e. MR-positive. After further incubation Escherichia coli cultures produce even more acid and in spite of phosphate buffer in the medium may be self-sterilizing; the MR test remains positive. Cultures of Klebsiella pneumoniae subsp. aerogenes, on the other hand, decarboxylate and condense the pyruvic acid to form acetylmethylcarbinol, the pH value rises and, when methyl red is added, the colour is yellow, i.e. MR-negative. Nowadays there is a tendency to do biochemical tests earlier but the temptation to speed up the MR test should be resisted; it should never be read until the cultures have been incubated for at least 2 days at 37 °C or 3 days at 30 °C. The reaction cannot be accelerated by increasing the glucose content of the medium; Clark & Lubs (1915) found that, in media with much above 1% glucose, cultures of 'K. aerogenes' (K. pneumoniae subsp. aerogenes) did not revert to become MR-negative. Despite its simplicity, we suspect that the MR test is rarely used in clinical laboratories. 37

BACTERIAL CHARACTERS AND CHARACTERIZATION 4.5.29 Milk (usually as Litmus Milk or Bromcresol Purple Milk) is a good nutrient medium in which most organisms will grow and it has a fairly constant composition since man only interferes by removing the cream and adding an indicator. Although highly esteemed elsewhere, in the medical laboratory Litmus (or Bromcresol Purple) Milk occupies a secondary position and most bacteriologists believe that the information it gives can be obtained with more certainty by using other media; for this reason, we do not use it in the tables. An objection to milk is that unless a change takes place in the appearance of the medium (e.g. acid or clot formation) it is difficult to be sure that growth has occurred. Milk contains lactose, galactose, a trace of glucose, casein, and mineral salts. Acid production from the fermentation of lactose is shown by a change in colour of the indicator, and, when much acid is produced, by the formation of a clot. But another form of clot may be produced by rennet; in this case the clot forms first and later, like the fibrin clot in blood, contracts and expresses a clear whey. In contrast the acid clot does not contract. When the bacterium also produces proteolytic enzymes the clot may be peptonized. Apart from the rennet clot (for which milk is a unique medium) all the other reactions can be detected more easily by using media appropriate for each reaction. Caseinase activity may be detected in a solid medium by incorporating skimmed milk in a nutrient agar base. Proteolysis is shown by the development of clear zones in the medium around areas of growth. Alternatively, substitution of soluble casein for milk gives a clear medium in which casein hydrolysis is seen as a clear zone around growth after the plate is flooded with 30% trichloracetic acid or with acid mercuric chloride solution (cf. Frazier's gelatinase test, Appendix C3.23). 4.5.30 Niacin (nicotinic acid) production is a feature characteristic of human tubercle bacilli and it distinguishes them from bovine tubercle bacilli and other mycobacteria (Pope & Smith, 1946). Several modifications of the test method have been devised; all are based on the extraction of niacin from the bacterial growth and subsequent detection by a colorimetric reaction. If used, the test must be done with care not only because of infection risks but also 38

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because one of the reagents (cyanogen bromide) is lachrymatory and toxic. 4.5.31 Nitrate reduction may be shown either by detecting the presence of one of the breakdown products, or by showing the disappearance of nitrate from the medium. The products of reduction may include nitrite, hyponitrite, hydroxylamine, ammonia, nitrous oxide, or gaseous nitrogen. The first test to be applied aims at showing the presence of nitrite. When this test is negative (i.e. nitrite is not detected) the medium is tested to see whether there is residual nitrate; if this test also is negative it confirms that the first stage of the breakdown has been completed and the nitrite further broken down. In uninoculated Nitrate Broth and with cultures of organisms that do not reduce nitrate, the test for nitrite is negative until zinc dust (ZoBell, 1932) or other reducing agent is added to the culture medium to reduce the nitrate contained in it. To detect small amounts of residual nitrate the amount of zinc added may be critical (Steel & Fisher, 1961). The tests are very sensitive and it is important to check the uninoculated medium for nitrite, which should not be present. Some workers prefer to carry out the test in a semisolid medium (ZoBell, 1932); others insist that free access to oxygen is necessary for nitrate reduction by aerobes. Conn (1936) discussed the difficulty of recording the results of the nitrate reduction test, and advised that the terms positive and negative be avoided. Instead the actual finding(s) should be recorded; the possibilities are shown in Table 4.1. An entirely different method was described by Cook (1950) who found that when nitrate was included in Blood Agar base, nitrate-reducing bacteria growing on the Blood Agar medium reduced the haemoglobin to methaemoglobin; this method has the advantage that the change seen is apparent even when the organism can also reduce nitrite. A convenient alternative way of performing the test is to apply a blotting paper disc impregnated with potassium nitrate on an inoculated plate of plain Blood Agar. 4.5.32 Nitrite reduction can be brought about by certain bacteria incapable of reducing nitrate (ZoBell, 1932). It can be shown by growing the organism in a

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SECONDARY TESTS USED IN THE TABLES

Table 4.1 Interpretation of tests for reduction of nitrate and nitrite Culture medium

Test applied

Nitrate Broth

1 For nitrite 2 For nitrite 3 Zinc dust

Interpretation

Result Colour not changed (negative) Red colour (positive) (A) Colour not changed (B) Red colour

Nitrite Broth

4 For nitrite

(C) Red colour (D) Colour not changed

broth containing 0.01% NaNO2 and, after sufficient time for the reduction to take place, testing for residual nitrite (Table 4.1). 4.5.33 The nitrogen nutrition of bacteria can be studied in a mineral base with a chelating agent (Owens & Keddie, 1969). The method has considerable interest in classification work but the standards of chemical cleanliness required are higher than some bacteriological laboratories can meet. But this should not deter anyone from trying the method. 4.5.34 O/129 sensitivity. The pteridine derivative O/129 (Appendix C1.18; C3.41) was regarded as almost specific in inhibiting the growth of vibrios, although the number of strains showing resistance to this agent seems to be increasing (Matsushita, Kudoh & Ohashi, 1984; Gerbaud et al., 1985). However, it is still a useful differential test to apply to any non-fluorescent, oxidase-positive, Gram-negative rod. Simple methods, such as drops of a 10% suspension on an inoculated plate of Nutrient Agar containing 0.5-1% NaCl are adequate though dried discs containing 150 L | Lg of this 'vibriostatic agent' may give more reproducible results; discs containing 10 |ig have some differential value within the genus. The discs can be prepared in the laboratory or obtained commercially (Lee & Donovan, 1985). 4.5.35 noside) menters days to

The ONPG (o-nitrophenyl-8-D-galactopyratest is used to detect potential lactose ferwhich, in ordinary media, either take several produce acid or do not produce any acid.

Nitrite not present (see Test 3) Nitrate reduced to nitrite Nitrite not present: therefore nitrate in original medium has been reduced completely by the bacteria Nitrate in medium reduced to nitrite by zinc but not by bacteria Nitrite present; not (all) reduced Nitrite reduced by bacteria and has disappeared from the medium

Lactose fermentation depends on two enzymes, (i) an induced intracellular enzyme, p-galactosidase, which attacks lactose, and (ii) a permease which regulates penetration of the lactose through the cell wall. Kriebel (1934) found that late lactose-fermenters produced acid more quickly when the concentration of lactose was increased to 5%; Chilton & Fulton (1946) recommended 10% lactose in agar. However, the results of the 5 and 10% lactose tests and the ONPG test for |3-galactosidase do not always agree (Lapage, Efstratiou & Hill, 1973). Lederberg (1950) used ONPG for the study of P-galactosidase, and Le Minor & Ben Hamida (1962) developed a rapid ONPG test for toluene-treated bacterial cultures. Lowe (1962) found that toluene treatment was not essential to liberate the p-galactosidase and that overnight incubation of cultures in peptone water containing ONPG hydrolysed the colourless substrate to the yellow onitrophenol. 4.5.36 Phenylalanine can be converted by oxidative deamination to phenylpyruvic acid (PPA) which, like many other keto acids, can be identified by adding ferric chloride (Singer & Volcani, 1955). The phenylalanine test was used by Henriksen & Closs (1938) who found that Proteus species gave the strongest reactions but 'Klebsiella aerogenes' (K. pneumoniae subsp. aerogenes) also gave some positive results. Since then Henriksen (1950) and others (Buttiaux et aL, 1954; Singer & Bar-Chay, 1954; Shaw & Clarke, 1955) have found it to be almost specific for Proteus, Morganella and Providencia. This specificity prompted Singer & Bar-Chay to put the 39

BACTERIAL CHARACTERS AND CHARACTERIZATION Providence organisms into the genus Proteus; however, the two genera are now recognized as distinct. The phenylalanine deaminase of Moraxella phenylpyruvica seems to be weaker than that of Proteus species and the usual methods do not work well with it. Snell & Davey (1971) describe a method in which the tubes are agitated at 37 °C and give positive results in about an hour. 4.5.37 Phosphatase activity was used by Barber & Kuper (1951) to aid the identification of pathogenic staphylococci; they found a high degree of correlation between phosphatase and coagulase production. By prolonging the incubation period, Baird-Parker (1963) demonstrated phosphatase production in 378 of 546 strains of staphylococci and 10 of 677 strains of micrococci. Some workers prefer a liquid medium and Lewis (1961) compared the plate and tube methods; he found that essentially similar results were obtainable in a liquid medium incubated for 6 hours and on a solid medium incubated for 18 hours with coagulase-positive staphylococci, but the tube method showed far fewer phosphatase-positive coagulase-negative strains. Among enteric bacteria, Voros et al. (1961) found that phosphatase was produced only by strains of Proteus and Providencia; using the same technique Cowan & Steel (1974) were unable to confirm this specificity and found positive reactions also in the Salmonella, Shigella, Klebsiella and Escherichia groups; for this reason, we no longer include the phosphatase test in the tables for identification of enterobacteria in this Manual. 4.5.38 Pigment formation often has considerable diagnostic value and it is an advantage to know how to encourage it. Although the pigments produced are seldom photosynthetic, most bacteria dealt with in this Manual form pigment better in the light; this is most noticeable in the staphylococci and serratias, but also occurs in the pseudomonads and in chromobacteria. The effect of light on pigment production by mycobacteria has become a means of distinguishing species. Temperature and medium also influence the intensity of pigmentation; most bacteria produce pigments better at temperatures below the optimum for growth, such as 22 °C for mesophils. Medium probably has the biggest effect on the development of pigment. In some cases the simple 40

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addition of glucose will enhance pigmentation, in other cases this will inhibit it. The old adage that 'one man's meat is another man's poison' applies also to bacterial pigment production and different formulae are needed for different organisms. The elimination of all meat extracts and the addition of mannitol are beneficial for Chromobacterium and Janthinobacterium and may improve pigmentation of Serratia strains (Goldsworthy & Still, 1936, 1938); quite different media are needed to encourage pigmentation by pseudomonads (see Appendix A2.4). On routine laboratory media, some strains of Pseudomonas cepacia produce yellow or brown non-fluorescent pigments which are soluble in water and chloroform. On chemically defined media, the pigments may exhibit a wide variety of colours depending on the carbon source used for growth (Lennette et al., 1982). The characteristic black pigment produced by some strains of the melaninogenicus-oralis group of Bacteroides during growth on media containing blood is due to the formation of protohaemin, and the red fluorescence of their colonies in ultraviolet light is due to protoporphoryn, a precursor of protohaemin (Shah et al., 1979). Yellow-green fluorescence in long-wave ultraviolet light is also exhibited by colonies of Clostridium difficile. 4.5.39 Poly-p-hydroxybutyric acid (PHB) may accumulate as a cellular reserve material; it was used by Stanier, Palleroni & Doudoroff (1966) in their characterization of Pseudomonas species. The chemical method of extracting and identifying this polymer (Williamson & Wilkinson, 1958) is not suitable for routine use, but examination of wet preparations of the organism by phase-contrast microscopy or of films stained by weak carbol fuchsin will reveal the intracellular deposits. PHB is produced most abundantly when the organism is grown in a medium containing D, L-P-hydroxybutyrate; after growth, films are stained with Sudan black and counterstained with safranin to reveal the purple-black deposits of PHB A within cells. 4.5.40 Survival under certain adverse conditions (usually heat) may have diagnostic significance; for example, streptococci that survives heating at 60 °C for 30 minutes (and which form the basis of the recently described genus Enterococcus) are likely to

SECONDARY TESTS USED IN THE TABLES

belong to Lancefield Group D. The tests themselves are not easy to standardize and the methods used by different authors vary greatly. For example, after the heating test for Enterococcus (Streptococcus) faecalis an immediate subculture may fail to show growth, whereas if the heated broth is incubated overnight, a subculture is more likely to yield growth. Other factors may affect the result, including the nature of the medium or suspending fluid in which the heating is carried out, its pH value, the time allowed for the medium to heat up to the desired temperature, and the type of container, particularly the thickness of the glass, in which the sample is heated. Some authors (Abd-el-Malek & Gibson, 1948a) always used milk, which they regarded as a medium of more constant composition than man-made infusions and enzymic digests. While the testing of vegetative bacteria for survival has its difficulties, the testing of spore suspensions is even more full of pitfalls. The heat stability of spores not only varies from one species to another, but even in the same species will vary from strain to strain, and spores of trie same strain grown on different occasions do not necessarily have the same resistance to heat. For a discussion of this subject the reader is referred to papers by Kelsey (1958, 1961). 'Heatshock' of bacterial spore suspensions is a useful technique employed to initiate germination. The resistance of spores to ethanol is used in the so-called 'alcohol-shock' treatment, which also destroys the accompanying vegetative forms. 4.5.41 Temperature range for growth and the optimal growth temperature are characteristic of different groups of bacteria; of those in the medical and veterinary fields the optimal temperature is usually between 35 and 40 °Cbut the range for growth varies considerably. Some species (e.g. Neisseria gonorrhoeae) have only a narrow range and rapidly die at temperatures outside the range; other organisms have a wide growth and an even wider survival range. In all cases the optimal temperature for growth is near the maximum of the temperature range. Biochemical tests are usually made on cultures grown at the temperature optimal for growth; however, this may not be optimal for the development of the product to be tested. For example, acetoin production by hafnias occurs at a lower temperature than the

[4.5] growth optimum. Some salmonellas are able to grow on a medium containing an ammonium salt as nitrogen source at 30 °C but will not grow on this medium at 37 °C,the optimal temperature for growth on media providing organic nitrogen. The ability of an organism to grow at 20-22, 30, and 37 °C are tested in many laboratories and the diagnostic tables show, in general, the more specialized tests used for different groups of bacteria; for these, adjustable water baths or incubators are needed. In the differentiation of species of Mycobacterium (Table 6.10/?) growth or survival is shown at several different temperatures. 4.5.42 Temperature tolerance. Most mesophilic bacteria in the vegetative state are killed at 56 °C for 30 minutes. A few species such as Staphylococcus aureus, Aerococcus viridans, and some streptococci (mostly belonging to the genus Enterococcus) survive heating at 60 °C for 30 minutes; this degree of heat-tolerance can be used as a screening test for them. 4.5.43 Tween 80 test for lipolytic activity (Sierra, 1957). Tween 80 is the oleic acid ester of a polyoxyalkylene derivative of sorbitan. It can be included in a suitable nutrient medium and the test culture(s) streak-inoculated on the surface; after incubation at the optimal temperature for the organism(s) under test, the plate is examined for opaque haloes around the growth. The opacity, which indicates lipolytic activity, is due to crystal formation. Tween 80 can also be incorporated in a liquid medium, which is used for the much longer period of incubation (up to 21 days) needed by mycobacteria (Kubica & Dye, 1967). 4.5.44 Urease activity is tested in Christensen's (1946) urea medium which supports the growth of many bacteria. The urease activity of Proteus, Providencia and some Klebsiella species can be shown in a highly buffered urea medium (Stuart, van Stratum & Rustigian, 1945) in which other enterobacteria appear to be urease negative. Proteus species can use urea nitrogen but most other ureaseproducing organisms need an additional nitrogen source for growth. Urease activity provides a useful test for distinguishing between Clostridium bifermen41

BACTERIAL CHARACTERS AND CHARACTERIZATION tans and Clostridium sordelli. Urease activity is shown by alkali production from urea solutions, but, in at least two methods (Elek, 1948; Ortali & Samarani, 1955), Nessler's reagent is added to show the presence of ammonia. 4.6

Rapid methods for the screening and identification of bacteria

Rapid, multitest micromethods - manual and mechanized - have now become firmly established in microbiological practice. Many of the early methods relied upon heavy inocula with preformed constitutive enzymes to demonstrate biochemical activity in miniaturized conventional methods (Clarke & Cowan, 1952). The majority of micromethods were developed for the Enterobacteriaceae, but more recently special methods have been introduced for the non-fermenters, anaerobes, streptococci and staphylococci. The pioneering work of Buissiere & Nardon (1968) on single substrate multi-test methods from which the API system evolved has led also to the development of several off-the-shelf kits with special plastic strips for single-step procedures for bacterial identification. The current emphasis is for reproducibility, speed, standardization and ease of use. Other microtest identification systems with 96-well microplates have been developed both commercially and by individual laboratories, as have multiple inoculation procedures for agar plates containing different substrates. Paper discs impregnated with substrates have also been used. All these techniques depend on a definitive result within 48 hours of inoculation and many can now provide presumptive identifications within 4 hours. The standard of quality control by manufacturers is higher than most individual laboratories can provide: we do not think, for example, that many laboratories could carry out melting-point analyses on their test substrates before the medium is prepared. The use of numerical taxonomic techniques, first described by Sneath (19572?) and discussed more fully in Chapter 8, has become so refined that data derived from such studies can now be used for computer-assisted identification of bacterial strains. Many commercial kits utilize such databases and several different procedures are available for interpreting the 42

[4.5]

patterns of results. Most commercial kit manufacturers provide indices of coded test-result patterns or profiles in numerical order which give a primary level of identification; for further help, many manufacturers operate telephone desks for direct assistance usually with the aid of a computer. The indices are normally prepared by combining test results to form octal sets as described in Chapter 10. 4.6.1 Manual biochemical systems. We do not intend in this section to review all the commercially available identification and screening kits, but rather to illustrate the methodology used with examples of kits currently available in the United Kingdom. We will describe the overall principles but not the specific methods as these may be found in the product literature issued with each kit; new commercial kits are launched frequently and clinical evaluations of them are usually published in scientific journals such as the Journal of Clinical Microbiology. There are two varieties of manual micromethods: (i) dehydrated substrates contained in plastic wells or compartments or on absorbent paper discs or strips, and (ii) multiple conventional substrates incorporated in agar for inoculation with one or more organisms. 4.6.1.1 Dehydrated substrates. The combination of several dehydrated substrates in one package or kit allows for 'unitary' identification, where all the tests are inoculated or the results interpreted in one step. Certain impregnated paper discs, such as those for indole or oxidase, are also available as individual conventional tests. For use, all kits with dehydrated substrates are inoculated with suspensions of the test organisms; as the required density of organisms may vary according to the kit, the suspensions should be prepared as described by each manufacturer. We make no apology for reminding users of this Manual that the bacterial cultures for identification must be pure. In the Minitek system individual impregnated paper discs from a wide range can be combined in a plastic tray. Some of the tests need a layer of sterile mineral oil or paraffin to ensure an adequate microaerophilic reaction; and after incubation for 18-24 hours, some tests require the addition of reagents. To use the manufacturer's Index of the most likely sets of results for the Enterobacteriaceae, 14

RAPID METHODS FOR THE SCREENING AND IDENTIFICATION OF BACTERIA

specified tests must be used. This system has not proved popular in the United Kingdom for several reasons, including: the freedom of test choice; the fact that a set of 14 tests is insufficient for a reasonable identification level for the Enterobacteriaceae; and the plethora of other kits available at similar cost. A 4 hour test version is now available but requires heavier inocula. The system can be used for the identification of anaerobes by choosing an appropriate set of substrate discs and incubation conditions appropriate for anaerobic identification. The Micro-ID system depends on the presence of preformed constitutive enzymes in the bacterial suspensions and so allows identification of the Enterobacteriaceae in only 4 hours. Each kit consists of 15 substrates dehydrated on paper discs in compartments in one plastic tray; the compartments hold both substrate and reagent discs. The test reactions are recorded after 4 hours (in one test after the addition of KOH) as a 5-digit octal profile. This can be interpreted by reference to the manufacturer's Profile Index. The system does permit some flexibility in recording the test results as the kits may be refrigerated overnight before final interpretation: trays inoculated later in the day can thus be placed in the refrigerator for reading the following morning. API have a wide range of kits available for many groups of medical bacteria. The API20E kit was designed for identifying enterobacteria, and consists of 20 special compartments ('Ivan Hall tubes') in a plastic strip. After incubation for 18-24 hours and the addition of appropriate reagents for the tryptophan deaminase, indole, and VP tests, it provides a 7-digit octal set of results or profile which can be looked-up in the Profile Index or referred to the manufacturer for computer-assisted identification. Since its introduction in the 1970s it has become one of the most popular identification kits with a good reputation for reproducibility and reliability as well as a worldwide database. Indeed the API50 test kits are currently regarded as one of the most convenient means for the identification of Bacillus species (Berkeley et al., 1984). The advent of plastic 96-well microtitre trays for serological analysis provided scope for other kits with potential for some degree of automated reading. Manufacturers have opted in most instances for a combination approach either by making part of the

[4.6]

tray available for antibiotic sensitivity tests or by subdividing the tray to allow several organisms to be examined simultaneously, e.g. 4 sets of 24 or 3 sets of 32 tests. For these kits, further manufacturing technology was required to ensure not only that the media remained at the bottoms of the wells when dehydrated but, more importantly, that they rehydrated uniformly when the bacterial suspension was added. 4.6.1.2 Agar-based identification systems. These identification systems fall into two groups: those in which one inoculation serves for all the tests for one organism, and those which require multiple inoculations of several media with several organisms. The principal drawback with these systems is the limited shelf-life of the media as, with prolonged storage and evaporation, some concentration of the constituent materials inevitably occurs. The Enterotube (Roche) is useful in circumstances where the full paraphernalia of a laboratory is not always available. A single plastic compartmentalized tube is divided into 11 chambers each containing media with single or multiple substrates. A thin metal rod runs through the base of each chamber; inoculation is achieved by touching a single colony on a solid medium with one end of the rod and pulling it through the whole tube, thus inoculating the medium in each chamber. The rod is then re-inserted to block the passage between the chambers and so ensure that no cross-contamination occurs between them. Reagents are added after overnight incubation and colour changes observed and recorded. Kits are available for both the fermenting and non-fermenting Gram-negative rods although only the former are currently regarded as satisfactory. A variety of multiple agar media in differing commercial formats have been marketed with varying success. One system used a multitest medium in a single tube followed later by a circular plastic mould with several compartments, each of which had to be stab-inoculated. These all required considerable manipulation at the inoculation stage and because of this drawback have not proved widely acceptable. Multi-point inoculation techniques for identifying bacteria have been available for many years. A commercial version (Cathra Replireader), marketed in the later 1970s, consisted of several agar plates with different substrates together with a replicate-inoculation 43

BACTERIAL CHARACTERS AND CHARACTERIZATION device for suspensions of multiple organisms for identification. A maximum of 36 isolates could be inoculated onto each of the 17 test plates for identification; extra plates containing antibiotics for sensitivity testing were also available. The method proved efficient and economic (Baer & Washington, 1972; Waterworth, 1980) but has failed to gain widespread acceptance principally because of quality control and supply problems with the commercially prepared agar plates incorporating the identification substrates. However, this kind of rapid methodology has reemerged commercially following the recent availability of accurate automated volume-control systems for the manufacture of packaged substrates for bacterial identification and antibiotic sensitivity purposes. An increasingly wide range of agar-based substrates has become available in tablet form (Mast) and this methodology, in association with multipoint agarbased antibiotic sensitivity testing, has proved convenient, flexible, efficient and economical. Various schemes to provide computerized databases have been proposed but because of the variety of different media and suppliers we think it unlikely that any single method will take precedence. Rather, we suspect that the trend will be towards laboratories creating and supporting their own databases (Clayton et al., 1986). The principal advantage of this approach lies in effective and ongoing in-house quality control and assessment both of laboratory procedures and in the identification of organisms. Provided that the test reactions are monitored carefully in comparison with selected control organisms (see Appendix D) this approach will provide most of the advantages described by Clarke & Cowan in 1952. 4.6.2

Other rapid methods

4.6.2.1 Monoclonal antibodies. The recent introduction of monoclonal antibodies, produced from single cloned hybridoma B cells, has considerable potential for bacterial identification purposes. As each B cell line produces a single immunoglobulin the antibodies have become known popularly as monoclonals. More details about the production and diagnostic use of monoclonal antibodies can be obtained from the publications of Kohler & Milstein (1975), Nowinski et al. (1983) and McLauchlin et al. (1988). Direct tests are becoming increasingly available 44

[4.6]

for many bacterial antigens and they have an enormous potential in rapid, definitive microbiology not only in the diagnostic laboratory but also at the bedside or in the surgery. Indeed commercially available kits to detect Group A streptococci in throat swabs within minutes are already available and we think that this area of rapid methodology is likely to develop considerably. 4.6.2.2 DNA probes. This recently developed technique depends on the natural process whereby single-stranded DNA is attracted to complementary strands to form double-stranded hybrids. Singlestranded, radiolabelled bacterial DNA (the probe) can thus be used to seek out complementary DNA strands in material such as culture preparations, bacterial colonies and even clinical specimens directly. The procedure is usually carried out on a solid phase such as a nitrocellulase filter; double-stranded DNA hybrids or duplexes remain bound to the filter while the single-stranded, unbound DNA is washed off. The technique has numerous potential applications and could be used, for example, to rapidly screen large numbers of samples effectively and economically. The following references will provide the reader who wishes to explore this technique with a few leads: Escherichia coll (Moseley et al., 1982; Romick, Lindsay & Busta, 1987), Neisseria (Totten et al., 1983) and Mycobacterium (Cooper et al., 1989). 4.6.3 Mechanized or automated methods. In this section we first discuss screening methods which help with the handling of clinical specimens, and then methods that assist with the identification of bacteria isolated from them. As before, we intend this as an introduction to the principles and the technology rather than as a review of every method. Evaluations and descriptions of the latest methods are usually published in scientific journals such as the Journal of Clinical Microbiology. Mechanized methods have been used in many clinical laboratories particularly outside the UK. The majority of these systems utilize micromethods with in addition automated interpretation of test reactions. The principal drawbacks are the high capital and revenue costs, especially for the examination of relatively few specimens.

RAPID METHODS FOR THE SCREENING AND IDENTIFICATION OF BACTERIA

4.6.3.1 Screening methods. The ability to detect the presence of potential pathogens in a clinical sample with a semi-automated method would be a boon to all laboratories: the advantages of processing more than 1 000 specimens per day are obvious. Several methods are commercially available though only for a limited range of specimens, notably urine, CSF, sputum and blood cultures. Several mechanized screening methods for urine have been described. They include the measurement of optical densities after incubation of small samples in suitable growth media; the quantitative measurement of the luminescence of bacterial ATP; measurement of the relative uptake of certain dyes or of comparative fluorescence; and enumeration and measurement of the size of the particles in urine samples with modified blood cell counters. Each method has its devotees (and sceptics) and we make no judgement on their relative merits although we are sure that these procedures will develop and become more widespread in the future. The screening of blood cultures has proved a larger problem. The incorporation of special substrates into blood culture media has allowed the application of radioisotope and infrared methodology as well as uncomplicated, visual gas production methods. Much effort has been directed towards measuring the minute changes in electrical conductivity across defined media in the presence of rapidly growing bacteria, though effective control of the inherent variables has proved expensive to implement successfully. 4.6.3.2 Identification methods. Apart from manual methods which lend themselves to mechanized or automated interpretation, an array of specialized systems have been developed for the identification of pure cultures. Once again the enterobacteria have been the prime area of interest, although the Gram-negative non-fermenters have an appreciable following.

[4.6]

The instruments available are based on manual methods and many still use the same test reactions but with specially developed substrates in plastic cartridges or cuvettes suitable for automated reading in photometers. The more expensive instruments incorporate microcomputers which analyse the reaction patterns and provide a computer-assisted identification of each test isolate. The majority of the instruments are capable of providing accurate levels of comparative identification. However, published evaluations often fail to stress the basic microbiological concepts discussed in Section 5.2, and we cannot stress too strongly again that pure cultures are imperative. 4.6.4 General aspects. Although microbiological mechanization and rapid methodology have relatively high capital costs and revenue consequences, other factors should also be considered. The shorter time from isolation to identification is an obvious advantage provided it can be completed within a normal working day. The increased sensitivity and specificity derived from automation and strictly controlled manufacturing conditions should provide microbiologists with firmer information upon which to base clinical judgement. We do not think, however, that the basic principles of microbiology will be understood so easily by the next generations of microbiologists if in future the materials and methods are simply to be taken from the refrigerator or shelf as kits. We believe strongly that time-honoured media and methods have an important part to play in training microbiologists. We wonder also, when instruments break down (as they will inevitably), whether the expertise of traditional procedures will be available? It seems to us that diagnostic microbiology is at a turning point and the new technology route should be followed with some caution.

45

Theory and practice of bacterial identification

It goes without saying that adequate clinical information is essential to enable appropriate culture media to be chosen for primary isolation. We believe that the subsequent investigation of the organism(s) thus isolated should be approached with an open mind; the organism(s) should be regarded as unknown and the process of identification started from the basic (primary) characters. 5.1

Theory of identification

In theory the identification of a bacterium consists of a comparison of the unknown with the known, the object being the ability to say that the unknown is like A (one of the known bacteria) and unlike B-Z (all other known bacteria); another and arguably equally important objective is to be able to say that the unknown organism is the same as A and thus to give it a name or identification tag. When we say that it is A we imply that it is different from all the other known bacteria, B-Z. All identification schemes depend on knowing a great deal about the already identified (or known) units, but the human memory can cope with only a small proportion of this knowledge, so memory aids are essential. In practice there are at present two distinct methods of making the identification; but the feasibility of a third method using a computer, first suggested by Payne (1963), was demonstrated by Dybowski & Franklin (1968) and is discussed in Chapter 10. The first method is familiar to all biologists and uses the dichotomous key. Characters are taken in turn and the keys are most successful when the features can be expressed unequivocally as either positive or negative. Although Streptomyces species are not described in this Manual, Kiister's (1972) claim that dichotomous keys are the most workable for the 46

classification and identification of that genus deserves notice. In choosing the sequence of characters he tried to take first those that were easiest to determine, and he followed the same sequence for all subdivisions of the genus. Kuster's scheme used seven characters which he was able to determine from observations made of cultures grown on only two media. With this economy of effort he (i) made primary groupings from the colour of the aerial mycelium, and (ii) subdivided these groups by the presence or absence of a distinctive pigment on the reverse side of the vegetative mycelium. The other characters used were, in order (iii) melanin reaction, (iv) formation of soluble pigment, (v) morphology of the spore-producing/bearing structures (sporophores), (vi) morphology of spores, and (vii) further subdivisions made on the utilization of one or more carbohydrates. The only dichotomous key to deal comprehensively with bacteria is that developed by Skerman (1949, 1959-67) and the successive versions have been increasingly useful. Another form of dichotomous key, the flow chart, can make allowance for the variable reactions given by strains of some bacterial species as in that prepared by Manclark & Pickett (1961). Thus, in this Manual what we call a 'd' character (different reactions in different strains, positive in some, negative in others) is treated in the flow chart as both positive and negative, and the species appears in at least two places. Tables make up the second memory aid, and these are widely used in all laboratories. It is easier to see the essential characters in a table than in pages of descriptive matter, which is seldom precise and often made unnecessarily vague by phrases such as 'most strains are...', 'some strains do not../, 'not infrequently strains...', and the impossible 'strains showing no...'.

THEORY OF IDENTIFICATION

Table 5.1. Symbols used in Chapters 6 and 7 and equivalent descriptive terms Symbol + d () (d) w (w) w/D ?

Meaning and descriptive equivalent 85-100% strains are positive (all, most, many, usually) 16-84% strains positive (many, several, some) 0-15% strains positive (none, one, few, some) Delayed reaction in test or delayed growth Different reactions given by different strains; positive reactions often delayed Weak reaction or growth Reaction or growth delayed and weak Weak reaction or no reaction with different strains; positive reactions are weak or growth is feeble Different reactions given by lower taxa (genera, species, varieties) Not known or insufficient information Not applicable

Table 5.2. Additional symbols used in some tables in Chapters 6 and 7 Symbol A C F G H J M NT O [O] [0] R r S s T U V X Y VP

Meaning Aerobic atmosphere preferred Curved in shape Fermentative; fermentation Gas produced Helical or spiral in shape Generally positive in young cultures; inconstant in older cultures Microaerophilic conditions preferred Not testable Oxidative; oxidation Under aerobic conditions Under anaerobic conditions Rod-shaped (bacillus) Resistant (to antibiotic, etc) Sphere; coccus Susceptible; sensitive (to antibiotic, etc) Spores terminal Spores central Spores subterminal or central; variable in position Spores oval; ellipsoidal Spores round Voges-Proskauer test Letters in bold type in tables are serological designations. Superior italic letters are explained in footnotes to the individual tables.

The construction of diagnostic tables would be simplified if all strains of one species behaved alike, and if the results of all tests could be expressed

[5.1] unambiguously as either positive or negative. Unfortunately as neither of these is ever likely to happen, we are forced to use various symbols to indicate the constancy or inconstancy of bacterial characters. The symbols now in use were developed from those applied by Kauffmann, Edwards & Ewing, (1956) and later adopted in reports of the Enterobacteriaceae Subcommittee of what has become the International Committee on Systematic Bacteriology of the International Union (formerly Association) of Microbiological Societies (ICSB of IUMS). Neither Kauffmann nor the Subcommittee fixed any numerical values to the symbols and in the first edition of this Manual a small table was given showing the values used in preparing the diagnostic tables for that edition. In Table 5.1 we show the assessment of these values and the gradings used in Chapters 6 and 7 of this third edition; we also show the approximate equivalents of descriptive terms used for reactions or expressing the results of tests; these equivalents (which are subjective) are necessary because so few characterizations are expressed quantitatively. Few laboratories other than those with reference functions have examined a sufficient number of strains of the less common bacteria to make test results expressed as percentages meaningful; exceptions to this statement are the Centers for Disease Control, Atlanta, Georgia, USA and the National Collection of Type Cultures, London, UK which, over a period of years, have handled large numbers of cultures and contributed many publications in which the results were expressed quantitatively. The tables of Lennette et al. (1985) for a large range of bacteria, and those of Edwards & Ewing (1962, 1972) and Ewing (1986) for a more limited range, are particularly valuable. Some characters are almost invariably positive or negative; unfortunately characters of such constancy are usually shared by similar organisms, and although they are important for characterization (and may appear in the miniature definitions given in Chapters 6 and 7), they have little value in distinguishing an organism from its neighbours, and thus seldom appear in the second-stage tables. The tables can form the basis of a set of diagnostic punched cards to be used with similar cards on which the characters of the unknown organisms are punched. Sorting the cards of the unknowns with 47

THEORY AND PRACTICE OF BACTERIAL IDENTIFICATION

those of the knowns can be one of the quickest, most accurate and least burdensome ways of arriving at the identification of an organism (cf. Riddle et al. 1956). We describe the use of the diagnostic tables in such a punched card system of identification in Chapter 9. The diagnostic tables can also form the basis for computer-assisted identification, which we discuss in Chapter 10. 5.2

Practice of identification

So far in this Manual we have discussed principles and indicated how all identification is based on a comparison of the organism we wish to identify with organisms of known identity. The accuracy of the identification depends on the thoroughness of the preparatory work such as media making, preparing stains and reagents, and the degree of care taken in carrying out, observing and recording the results of the various tests. In Chapter 3 we drew attention to the fact that bacteria isolated on inhibitory and selective media were likely to be mixed cultures, and we indicated some of the steps to be taken to purify a culture. It is not easy to be sure that such a 'purified' culture is incontrovertibly pure, and when there is any doubt whatever, it is safer and saves time to repeat the purification process. To identify a culture takes a great deal of effort and to suspect at the end that the culture may be impure is not only aggravating to the laboratory worker and the delay in receiving the report frustrating to the clinician, but it also indicates that the bacteriologist has wasted much time and material. Common organisms really are the commonest; and when an organism cannot be identified or seems to be an exotic species, we should consider the possibility that either our culture material is impure, or we have made some error in observation or recording. This happens to all of us and it reflects adversely on our ability and integrity when we fail to repeat observations, and go ahead believing that our results are infallible. There are various routes by which an identification can be arrived at; medical bacteriologists often have the advantage that they know what they are looking for, and at an early stage direct their investigation into certain special channels. This may sometimes turn out to be a disadvantage; for example, the 48

[5.1]

selective media used may inhibit the growth of a pathogen whose presence is unsuspected. Steel (1962a) discussed the different techniques used for identification of pure cultures. Basically there are three approaches to the problem; in the first, which we call the blunderbuss method, every conceivable test is done, and when all the results are available, the characters of the organism are compared with those listed in standard texts including Bergey's manuals of determinative and of systematic bacteriology. If all tests appropriate to the organism have been included, it should be possible to make the identification, but quite often other (possibly unheard of) characters are mentioned so that additional tests are needed; this is such a common experience that few bacteriologists follow the blunderbuss method. However, such a comprehensive investigation is necessary when the organism has to be characterized for its description as a new species. The second approach is based on probabilities and a judicious assessment of what kind of organism is causing the particular infective process. Thus, from a boil one would expect to isolate Staphylococcus aureus, or from the stools of a patient with an intestinal upset, one of the enterobacteria, and it would be reasonable to do tests that are likely to lead to as rapid an identification as is consistent with accuracy. When the most probable causal organism seem to be excluded, the investigator should continue with an open mind and follow the third approach. The third approach is the step-by-step or progressive method as used in this Manual, in which the first step aims at determining a few fundamental characters such as those used in Tables 6.1 and 7.1. When these characters are known (usually in 24 hours but occasionally needing 48 hours) another set of media can be inoculated to enable appropriate tests (given in second-stage tables) to be made; the number of these tests will always be less than that needed when the blunderbuss method is followed. Sometimes additional tests are needed for the better identification of a species, and these are shown in third-stage tables. In deciding what media to inoculate we are guided by the tests to be carried out, and we must decide for or against classical methods that are slow, as for example gelatin stab-cultures to show liquefaction or hydrolysis. Time can be saved by using multitest media in which several reactions can be observed at

PRACTICE OF IDENTIFICATION

one time; such methods are used mainly in the preliminary screening of large numbers of cultures, and they are useful in that 'non-pathogens' or organisms thought to be of low-grade pathogenicity can be detected and discarded without more ado, thus restricting further tests to those organisms that appear to fit into groups that contain potential pathogens. Other rapid methods may be considered: not only are they quicker than standard procedures but some, at least, give more clear-cut results; that of Clarke (1953a) for H2S production is, however, very sensitive and yields more positive results than are shown in the diagnostic tables. The range and use of rapid methods is discussed in Section 4.6. When all the tests have been completed the results are compared with the appropriate table(s); in this edition some species can be identified at the second stage, in others both the second- and third-stage tables should be consulted. For various reasons a species (or genus) may be shown in more than one

[5.2] table; we hope that these double entries will make identification easier for users of this Manual. In using the progressive tables in Chapters 6 and 7 it is important to remember that occasionally an organism of undoubted identity will have an anomalous character (such as a positive oxidase reaction in a strain of Salmonella typhi) so misleading that it will be impossible to make the identification from the tables. We have not made provision for exceptions such as this; neither have we made double entries for motile and non-motile variants of the same species. We would also remind those using Table 6.1 that asporogenous strains of Bacillus species can and do occur. Not all Bacillus species are Gram-positive but most of those likely to be isolated in medical laboratories are, and the genus is therefore described only in Chapter 6. However, Gemella, because its staining character (usually Gram-negative) can be misleading, is shown in tables in both Chapters 6 and 7.

49

Characters of Gram-positive bacteria

In characterization by stages, the first-stage table is combined with a figure and shows how, with a small number of selected characters, Gram-positive bacteria can be divided into groups that correspond to those used in orthodox classifications. Not all of the theoretically possible combinations of characters are shown in Table 6.1 because many of them do not seem to occur in nature. Each shaded square indicates the genus or genera that have the characters shown in the same column in the table above it. Equivocal characters, those difficult to determine, and characters markedly influenced by culture medium or test method can make a genus span more than one column; we have therefore tried to concentrate on the reactions given by most strains of a species in the kind of media likely to be used in routine diagnostic laboratories (majority reactions or characters) though in doing this we may perhaps have introduced a tidiness that is not warranted by the biological nature of the scheme. An example of generic spread is seen with Aerococcus, which appears in the third and fourth columns of Table 6.1; in this case, the reason for the spread is that the catalase reaction is not always easy to read and may be interpreted in different ways by different workers. Those who expect a large volume of gas to be produced may record the feeble reaction of A. viridans as negative whereas others, who habitually work with streptococci and are conversant with truly negative results in this test, will take more notice of the small bubble of gas that may be produced and record it as positive. Conflicting readings of this kind occurred when two laboratories co-operated in the work which led to the description of the new species Aerococcus viridans (Williams, Hirch & Cowan, 1953).

50

6.1

Division into major groups

As in previous editions of this Manual, the Grampositive bacteria are divided into several major groups, using the characters shown in the upper part of Table 6.1; they are shown as rectangles with broken lines and are numbered to correspond with the diagnostic tables in which further characterizing details are given. These major groups of bacteria are not accretions of related genera but are groups of convenience, groups of similarly shaped organisms, or groups of organisms that give similar results in the limited number of tests applied in the first stage of our identification scheme. Indeed, we emphasize that the groupings of the Gram-positive non-spore-forming rods encompassing diphtheroids (Section 6.5), actinomycetes (Section 6.7) and organisms intermediate between them (Section 6.6) are highly artificial. The classification of the bacteria in these groups has undergone marked changes in the past decade and is still not fully resolved (see Kandler & Weiss, 1986; Jones & Collins, 1986). The genus Corynebacterium, for example, is taxonomically close to Mycobacterium and Nocardia but, unlike them, corynebacteria are not acid-fast; since acid-fastness is an important identifying character in routine diagnostic laboratories, the corynebacteria are therefore grouped together with morphologically similar organisms as 'diphtheroids' in Section 6.5. The same considerations apply also to the Gram-negative bacteria which we deal with in Chapter 7 though they do include a 'group of difficult organisms' comprising miscellaneous bacteria that cannot reasonably be attached to other groups. Our intention in using major groups is to be strictly practical and accordingly the size of certain bacterial groups is determined to

[6.1]

DIVISION INTO MAJOR GROUPS

Table 6.1. First-stage table for Gram-positive bacteria 10 Shape Acid fast Spores Motility Growth in air Growth anaerobicallyt Catalase Oxidase Glucose (acid) Carbohydrates [F/O/-] Micrococcus a Staphylococcus Aerococcus Enterococcus Streptococcus Lactococcus Pediococcus b Gemella Anaerobic cocci * Kurthia Corynebacterium Listeria Brochothrix Erysipelothrix Lactobacillus Arcanobacterium Arachnia c Rothia Propionibacterium Actinomyces Bifidobacterium Eubacterium Clostridium d Bacillus Nocardia e Mycobacterium

* a b c d e t D d F 0 w ?_ [<>_] 1 4* 1

R

+

+

D +

+

—

d +

W

W

4-

4-

4-

-

VI-

-

— — — — +

w

D Ol-

+ F 6.2

R

R

11 R

12 R

13 R

14 R

15

16

R

R

-

-

V

VI-

4-

1+ 1

4

R

-

+ ? + F

F

17

+ ? -

R

D

18

19

20

R

R

R

D

+ D + ? d D D F/-F/O/-

+ + O

21

-

-

4-

+

_

9

+

4-

O O/NT

•

.....xz i

4* 4

6.3

4-

V

,6A 4

6.5

66

44* 4 4>

4* 4 4

6.7 <>

• <>

Peptococcus and Peptostreptococcus. Also Stomatococcus. Also Leuconostoc. Also Actinomyces odontolyticus. Exceptions: C. histolyticum; C. tertium; C. carnis. Also Actinomadura. Anaerobic growth of anaerobes inhibited by metronidazole. Different reactions in different species of the genus. Different reactions in different strains. Fermentation. Oxidation. Weak reaction. Not known. Asporogenous variants. Typical form.

^6.8

<> <>

<>

<>

^6.9

4 4>'

+ 1+ | 6.10

S R NT

Cultural characters of these organisms can be found in tables with the number indicated. Sphere (coccus). Rod-shaped (bacillus). Not testable.

Other symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

51

+'

CHARACTERS OF GRAM-POSITIVE BACTERIA

some extent by the size of tables that will fit into the page. We think that embracing the major groups in this way will help in identifying bacteria logically in stages or by steps. The consequences of the groupings can be seen in Chapter 9, which uses information from Tables 6.1 and 7.1 in a scheme of identification to the level of genus utilizing punched cards. The same information is used for the computerassisted bacterial identification programs described in Chapter 10. The bacterial groups are not named but colloquial or descriptive tags, such as anaerobic cocci and enterobacteria have been applicable to some of them. Some of the groups overlap, thus showing that they are not conventional taxa. Some characters that depend on biochemical and molecular biological techniques are very useful in bacterial classification but are not appropriate for day-to-day diagnostic work. Such characters include, for example, the chemical composition of the cell wall (of distinctive and particular value in the classification of Gram-positive bacteria), the lipid composition (including fatty acid profiles), isoprenoid quinone structural types, whole cell protein patterns and metabolic product profiles. These, together with the information derived from DNA-base ratios, DNA restriction patterns, DNA-DNA and DNA-rRNA homology values and rRNA oligonucleotide sequence cataloguing (see Goodfellow & Minnikin, 1985; Gottschalk, 1985) can be of great importance in classification but they are not characters that can be determined by simple tests. They do, however, play an important part in bacterial identification at the reference laboratory level. 6.2

The staphylococci and micrococci {Staphylococcus, Micrococcus, Stomatococcus, Aerococcus) Characters common to members of the group: Gram-positive cocci: aerobic. Catalase-positive; oxidase-negative (some exceptions). Indole and H2S not produced.

Grouping these genera of Gram-positive cocci together has been traditional though controversial for many years. At one time there were seemingly interminable arguments about Staphylococcus and Micrococcus, whether they should be separate or combined, and if combined what they should be 52

[6.1

named. Later, it was generally thought that only one group of catalase-positive Gram-positive cocci was justified, for which the name Staphylococcus was preferred by medical and Micrococcus by non-medical bacteriologists. By the time Cowan (1962/?) reviewed the situation, a distinction was made between those cocci that fermented glucose anaerobically (the staphylococci) and those that oxidized the sugar or did not produce acid from it (the micrococci). Other test results, including those for acetyl methylcarbinol (acetoin) and oxidase as well as sensitivity to lytic substances and serology, also favoured their separation. This has been strongly supported by numerical taxonomy (Hill, 1959; Feltham, 1979) and DNA studies (Silvestri & Hill, 1965; Kocur, Bergan and Mortensen, 1971) which clearly demonstrated significant differences in the GC base ratios between Staphylococcus (31-33%) and Micrococcus (69-75%); the recently created genus Stomatococcus (56-64%) is also clearly separate. Further confirmation of their validity as separate genera has come from studies of the structure of the cell wall (Schleifer & Kandler, 1972), and of components such as menaquinones (Jefferies et ah, 1968), fatty acids (Jantzen et ah, 1974) and fructose aldolases (Fischer et aL, 1983). More recently, sequencing of 16S ribosomal RNA has permitted the phylogeny of the genera to be determined (Schleifer and Stackebrandt, 1983). Indeed, Stackebrandt and Woese (1979) suggest that Staphylococcus is related to Bacillus but is a valid genus; Micrococcus, on the other hand, appears to be mixed with Arthrobacter in the coryneform group. In their view, Planococcus, a marine, singlespecies genus which is usually considered with staphylococci and micrococci, should be placed in the genus Bacillus together with Sporosarcina, but as these organisms are not pathogenic for man we mention them here only to dismiss them. Recent reviews on these topics include those of Goodfellow (1985, 1987), Alderson (1985) and Schleifer (1986). Table 6.2a shows the main distinguishing features between staphylococci and the micrococci. The genus Stomatococcus, described by Bergan & Kocur (1982) with a single species S. mucilaginosus, was previously included within Micrococcus. We show it separately in Table 6.2a but for convenience include it with the micrococci in Table 6.2c. We also include Aerococcus, the a group of Shaw, Stitt & Cowan

[6.2]

THE STAPHYLOCOCCI AND MICROCOCCI (1951) in these tables because it can cause confusion. This genus resembles more closely the streptococci (see Section 6.3) and can be distinguished readily from staphylococci by its failure to hydrolyse arginine and to reduce nitrate; we discuss it more fully in Section 6.3.2. The aerobic, packet-forming cocci previously referred to as the 'sarcinas' are now included in Micrococcus (Hubalek, 1969) thus limiting the genus Sarcina to anaerobic packet-forming cocci only (Shaw, Stitt & Cowan, 1951). The latter have been associated occasionally with postoperative complications of the genito-urinary tract. With such wide differences between Staphylococcus and Micrococcus it is surprising that it has proved so difficult to develop reliable differential characterization tests for strains of these genera. Several tests have been proposed though most of them are known to have exceptions (Baker, 1986). We list some of the differential tests in Table 6.2a and consider them here. The concept that staphylococci fermented glucose whereas micrococci oxidized it (or af least produced no acid from it) formed the basis for several variations of the Oxidation-Fermentation (O-F) test of Hugh & Leifson. However, as the results varied so much, a standardized test with a modified Baird-Parker (1963) medium was recommended for staphylococci (Subcommittee, 1965, 1976). Later, a thioglycollate broth test which obviated the need for a pH indicator and an oil seal was introduced (Kloos & Schleifer, 1975b); this test depends on the extent of growth in relation to the air/medium interface. Most staphylococci grow well throughout the broth medium though S. saprophyticus and similar strains appear to be oxidizing because they grow poorly in the anaerobic part of the tube; in contrast micrococci, except for M. kristinae which ferments glucose, grow only near the surface of the medium. Despite these exceptions, the ability to grow under anaerobic conditions is an important characteristic of Staphylococcus strains. The detection of cytochrome c in the oxidase test with 6% tetramethylphenylenediamine in dimethyl sulphoxide as the reagent (Faller & Schleifer, 1981) is characteristic of Micrococcus strains, although S. sciuri and S. caseolyticus are also positive. The more usual oxidase reagent is significantly less sensitive but still gives a positive reaction for most strains of

Table 6.2a. Second-stage table for Staphylococcus, Micrococcus, Stomatococcus and Aerococcus

Growth under anaerobic conditions Catalase Oxidase Carbohydrate attack VP Arginine hydrolysis Nitrate reduced Lysozyme Lysostaphin 1 2 3 4

Staphylococcus Micrococcus Stomatococcus Aerococcus

1

2

3

4

+ + — F + + +

+ d O/s r

+ w — F + + ? r

w w F -

r s

9 9

s == sensitive r == resistant

Other symbols used in the tables are explained in Tables 5.1 and 5.2 on p. 47.

Micrococcus (Boswell, Batstone & Mitchell, 1972). A positive result thus indicates that the organism is probably not a staphylococcus. The production of acetylmethyl carbinol (acetoin) from glucose in the Voges-Proskauer (VP) test has been used as a valuable character for separating the two genera (Kocur & Martinec, 1962). As the presence of phosphate can interfere with acetoin production (though not among enterobacteria; see Section 4.5.1) Baird-Parker (1963) emphasized the need to use a phosphate-free medium and to incubate for up to 14 days at 30 °C for staphylococci and micrococci. Under these conditions, all staphylococci except S. intermedius, S. hyicus, and S. simulans give a positive VP reaction whereas most micrococci are negative except for M. kristinae. It should be noted, however, that some commercial strip methods use pyruvate as the substrate and can therefore give results which differ from those with the Baird-Parker method (Marples & Richardson, 1982). The hydrolysis of arginine is also a useful test for separating staphylococci from micrococci (Peny & Buissiere, 1970; Feltham, 1979). Methodology is important but perhaps of more importance is the occurrence of arginine-negative strains of S. epidermidis (Marples & Richardson, 1981) and of argininepositive strains of Micrococcus (Marples & Richardson, 1980). 53

CHARACTERS OF GRAM-POSITIVE BACTERIA

The susceptibility of some Micrococcus species to lysozyme (Fleming, 1922) and of some Staphylococcus species to lysostaphin (Schindler & Schuhardt, 1964) as well as the greater resistance of staphylococci to erythromycin (Schleifer & Kloos, 19756) and bacitracin (Falk & Guering, 1983) and the resistance of micrococci to the nitrofurans (Curry & Borovian, 1976) have all been proposed as simple methods for separating strains of the two genera. These approaches may have some differential value but they are not definitive. Serology (slide agglutination) has also been used successfully for distinguishing between micrococci and coagulase-negative staphylococci (Nakhla, 1973). Although staphylococci are usually smaller than micrococci, neither this nor their Gram reaction are reliable criteria for their differentiation. Equally, pigmentation, though previously much stressed, is one of the least important characteristics of staphylococci; indeed non-pigmented (white) colony variants occur frequently. The pigmented 'violagabriellae' variant of S. epidermidis was regarded by Steel (1964) as an aberrant strain but Marples (1969) found that it occurred not uncommonly on human skin. Its reddish-purple pigmentation shows up well on simple media such as CYLG (Casein-yeast-lactate-glucose) or Potato Agar but is easily missed on media containing blood. Most staphylococcal pigments tend towards orange in colour whereas micrococcal pigments are typically greenish-yellow; but exceptions abound. Contrary to the usual belief, O'Connor, Willis & Smith (1966) did not find that exposure to daylight impaired pigmentation; Willis, O'Connor & Smith (1966) regarded fatty acids as more important and recommended a milk-cream agar. Despite the absence of a single wholly reliable routine laboratory test, we do not think that microbiologists experience much difficulty in correctly identifying a strain as a Staphylococcus or as a Micrococcus. For the selective isolation of staphylococci, particularly in food-poisoning investigations, Mannitol Salt Agar (Chapman, 1946), the egg-yolk tellurite medium of Baird-Parker (1962) and the potassium thiocyanate medium (SK) of Schleifer & Kramer (1980) are useful for inhibiting the growth of Gram-negative and other organisms; and Phenolphthalein-phosphate Agar may be used for the rapid detection of S. aureus colonies in mixed cultures (Barber & Kuper, 1951). 54

[6.2]

6.2.1 Staphylococcus (Tables 6.2a, b). For years, the genus Staphylococcus was regarded as virtually equivalent to Staphylococcus aureus, the main species pathogenic for man. The ability to coagulate citrated plasma virtually defined this potential pathogen and coagulase-negative staphylococci were disregarded. However, the taxonomy of this genus has undergone considerable revision, firstly in the recognition of phenotypes (Baird-Parker, 1963, 1965a, b) within the genus and, more recently, of species among the coagulase-negative human staphylococci (Schleifer & Kloos, 1975a; Kloos & Schleifer, 1975a, b) and secondly in the extension of taxonomic interest to animal and environmental staphylococci (Kloos, Schleifer & Smith, 1915a, b, 1976; Devriese et al., 1983). The potential pathogenicity of coagulase-negative staphylococci, particularly in urinary tract and foreign body infections, has also become accepted (Parker, 1981; Sewell era/., 1982). Some 26 species of staphylococci have now been validly described and we list these with their identifying characters in Table 6.2b. Species 1-14 are associated with man; the remainder are essentially animal strains though they may sometimes occur in opportunist infections in man. In man, S. aureus remains the predominant pathogen though S. saprophyticus can cause primary infections of the urinary tract (Hovelius, 1986). In addition, S. epidermidis, S. haemolyticus and S. capitis together with biotypes within S. hominis - such as the recently described S. lugdunensis (Freney et al., 1988) - are all able to act as opportunist pathogens (Parker, 1981). The few tests needed to differentiate all these species are marked with an asterisk (*) in Table 6.2b. Staphylococcal strains, particularly those of S. aureus, can produce a number of extracellular proteins which may have enzymic activities and may be toxic to tissues; although these products can have some differential value, they are not used in species identification and are not shown in the diagnostic tables. Some are recognized as haemolysins, as for example the a toxin, which is also the dermonecrotic toxin; this protein is antigenic and forms the basis for a clinical antibody test. The p-haemolysin is characteristic of animal strains of S. aureus though its production can be inhibited by the acquisition of temperate phages. Many enzymes have toxic effects on tis-

Table 6.2b. Third-stage table for Staphylococcus

G r o w t h anaerobically Oxidase *VP *Coagulase A c i d from Lactose *Maltose Mannitol Fructose Sucrose *Trehalose Xylose

1

2

3

4

5

6

7

8

+ _ + +

+ _ +

+ + + w w + a + w + + w w w + + w _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ + _ _ + + d ? + + + + + + + d , -

_ d -

+ + + + + + _

+ + + + + _

+ + D d + d -+ + + + + + + + + + + - _ _ _ _ _

d + + + + + +

d

9

-

D -+ — + + + d d + + + _ _ _

1 0 11 12 13 14 15 16 17 18 19 2 0 2 1 2 2 2 3 2 4 2 5 2 6

+ +

d ^ - d + + + + + + _ _

+ + + + + _

+ + + + + + _

+ + + +

+ + + + + + +

+ d + + + d + + + — + + + — + + + + — d + + + d d + + _ _ _ + +

9

Mannose *Phosphatase Nitrate *Arginine Urea Protease

*Novobiocin

+ + + + d

+ + + + +

+ + + + +

+ + + + +

+ D

+

+

s

s

s

s

+ + + + +

+ + + -

W

s

+ d ? d w d + + + + + + + ^ + ^ + d ? -+ + + +

W

s

s

-

-

s

-

s

1 Staphylococcus aureus; 6 Staphylococcus capitis S. pyogenes; Micrococcus 7 Staphylococcus auricularis 8 Staphylococcus saccharolyticus pyogenes var. aureus 2 Staphylococcus intermedius 9 Staphylococcus haemolyticus 3 Staphylococcus hyicus 10 Staphylococcus hominis 4 Staphylococcus chromogenes 11 Staphylococcus warned 5 Staphylococcus epidermidis; Staphylococcus saprophyticus; S. albus; Micrococcus pyogenes var. albus a b c d *

-

s

-

s 12 13 14 15 16 17

-

s

-

r

+ + + + - d + -

r

+ + + + +

-

-

-

r

s

s

Staphylococcus simulans Staphylococcus saprophyticus Staphylococcus cohnii Staphylococcus xylosus Staphylococcus caprae Staphylococcus carnosus

No growth anaerobically Usual reaction Ornithine decarboxylated Inferred reaction These tests are usually sufficient to identify the species that may infect man

Other symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

s = sensitive r = resistant

9

9

+ ? + ? -

? + ? ?

d + -

+

-

s 18 19 20 21 22 23

r

r

w _

d + + + + _

w -

w + -

+ + —— + + -

+ d + + + + _

+ + + + + _

+ - — + + + d _ _

_j_

_|_

_

+ + -c ?

_j.

_

+ + + +

+ + + ? +

+ ++ + ? d

d + + ? -

-

-

-

w

+

?

?

r

r

r

s

s

r

_

+ + + +

Staphylococcus caseolyticus 24 Staphylococcus sciuri Staphylococcus arlettae 25 Staphylococcus lugdunensis Staphylococcus equorum 26 Staphylococcus schleiferi Staphylococcus gallinarum Staphylococcus kloosii Staphylococcus lentus

CHARACTERS OF GRAM-POSITIVE BACTERIA

sues, including the PV leucocidin of Panton & Valentine (1932); fibrinolysin (Hajek & Marsalek, (1971); deoxyribonuclease (Elston & Fitch, 1964); phosphatase (Pennock & Huddy, 1967); coagulases (Cruickshank, 1937; Cadness-Graves et al, 1943); and the so-called a, p and 8 toxins or haemolysins. Some strains can also produce enterotoxins (A-E) with a powerful emetic effect in man and certain animals. Some strains of S. aureus (of phage group I) produce toxins which cause the 'toxic shock syndrome'; enterotoxin B has also been implicated in toxic shock (Schlievert, 1986). Many laboratories now screen isolates for DNase activity to identify them as presumptive pathogenic staphylococci. Of all these factors, however, the production of coagulases which can clot plasma is still a reliable and widely used test for the recognition of potentially pathogenic staphylococci, particularly those associated with acute infections. Two different coagulase methods can be used for presumptive pathogenic staphylococci: the tube test which detects 'free' coagulase and the slide test for 'bound' coagulase (also called the clumping factor). In tube tests, human strains produce coagulases which clot human and rabbit plasma but not always bovine plasma; conversely, bovine strains give a positive coagulase reaction more frequently with bovine than with human plasma. This test as well as the slide test for clumping factor ('pseudoagglutination' of staphylococci) are therefore best performed with rabbit plasma, which is commercially available; human plasma should be avoided unless it has been strictly controlled for clotting capability and absence of inhibitors. A variety of commercial slide agglutination screening tests are also available; some detect only the clumping factor but others also detect the staphylococcal cell-wall component 'protein A' which binds non-specifically to y globulins (Grov, Myklestad & Oeding, 1964). None of these slide tests is as reliable as a strictly controlled tube coagulase test (Dickson & Marples, 1986) but because of their ease of use and rapidity they are widely used as a good indicator of pathogenic potential. Taxonomically four main clusters or groups of species can be recognized respectively around S. aureus, S. epidermidis, S. saprophyticus and S. sciuri though the validity of all the 26 species is undeniable. The 'aureus' group of species includes S. aureus, 56

[6.2]

S. intermedius, S. hyicus, S. chromogenes and, more distantly, S. simulans. Although these species share several phenotypic characteristics (Feltham, 1979; Goodfellow et al., 1983), DNA hybridization clearly separates them (Schleifer, 1986). Although S. intermedius and S. hyicus cause infections in certain animals they have not been implicated yet in human disease. Strains of S. aureus can also be subdivided into a number of biotypes which reflect different sources. Meyer (1966) distinguished the 'hominis' from the bovis variety and Hayek & Marsalek (1971) further elaborated the differences, calling the hominis strains biotype A, some strains B, bovine strains C and other animal strains D, E & F. The E & F biotypes are now recognized as S. intermedius. Biotype A strains are associated with man; they usually produce fibrinolysin and the a haemolysin, and are susceptible to an internationally accepted set of phages for typing isolates from human infections (Asheshov & Rountree, 1975). Strains of bovine origin, biotype C, produce (3 haemolysin but not fibrinolysin and, except for phage 42D, they are susceptible only to special sets of phages developed for typing isolates from animals. Strains of S. intermedius and S. hyicus do not possess clumping factor for slide tests and they take longer to produce a clot in the tube test than human S. aureus isolates (Devriese et al., 1978). S. chromogenes and S. simulans are coagulase-negative. Acetoin production (VP test) is useful for differentiating S. aureus (positive) from the other coagulasepositive staphylococcal strains (negative). The 'epidermidis' group of species includes three subgroups: (i) S. epidermidis, S. capitis and some strains of S. warneri; (ii) S. hominis and the rest of S. warneri; (iii) S. haemolyticus, S. caprae and S. saccharolyticus. A distinguishing feature of the 'epidermidis' group is that almost all strains are sensitive to novobiocin in concentrations up to 1.6 |ig/ml in agar media (Schleifer & Kloos, 1975a). S. epidermidis, followed by S. haemolyticus, are the commonest coagulase-negative species associated with human infections other than those of the urinary tract (Nord et al., 1976; Marples & Richardson, 1981). S. epidermidis is relatively active biochemically but does not acidify mannitol or trehalose; 5. haemolyticus usually acidifies both these sugars. The

THE STAPHYLOCOCCI AND MICROCOCCI other species in the 'epidermidis' group are less active biochemically, less well defined and less likely to be associated with disease or to exhibit multiple resistance to antibiotics. Any of the species could be encountered as contaminants and occasional opportunist infections have been reported (Fleurette et aL9 1987). Taxonomic developments in this group should be expected. For S. epidermidis useful serological and phage typing schemes have been developed (Pillet & Orta, 1977; de Saxe et al., 1981). The 'saprophyticus' group of species includes S. saprophyticus, S. cohnii and S. xylosus. They are all resistant to novobiocin and exhibit anomalous resistance to penicillins and fusidic acid (Richardson & Marples, 1980). S. saprophyticus appears to act as a primary pathogen causing urinary tract infections in young women (Maskell, 1974) and possibly prostatitis and urethritis in men (Hovelius, 1986). The skin is probably the main source of these infections though the less pathogenic S. cohnii is the usual novobiocinresistant commensal species present in adult skin (Namavar et al., 1978). S. xylosus is an uncommon cause of human infection and most isolates are of animal origin (Marples & Richardson, 1981). The 'saprophyticus9 taxon is frequently used as a collective 'dump' in medical microbiology laboratories. The 'sciuri' group of species comprises S. sciuri and S. lentus, both of which are also novobiocinresistant. These species are of animal origin and differ from other staphylococci in giving a positive oxidase reaction and in their ability to ferment cellobiose. Numerical taxonomy (Feltham, 1979) and DNA hybridization (Schleifer, 1986) indicate that they are only distantly related to other species of Staphylococcus. However, some strains resembling S. sciuri and S. gallinarum have been isolated from human infections; such strains may give anomalous reactions in clumping factor tests. In summary, the staphylococci include the primary pathogens S. aureus and S. saprophyticus as well as the opportunist pathogens S. epidermidis and S. haemolyticus. The other species appear to be of lesser clinical significance though they may be involved, for example, in implant surgery and immunosuppressive therapy. In spite of the extended discussion about the genus Staphylococcus and its species, they are relatively easy to isolate and identify.

[6.2] Mini definition: Staphylococcus. Gram-positive cocci in clusters. Non-motile; nonsporing. Aerobic and facultatively anaerobic. Catalase-positive; usually oxidase-negative. Hydrolyse arginine; produce acetoin. Attack sugars by fermentation. Type species: S. aureus; NCTC strain 8532. 6.2.2 Micrococcus (Tables 6.2a, c). Hucker (1924&) originally included both staphylococci and micrococci in the genus Micrococcus but, as described in Section 6.2, opinions have since changed. Whether or not this varied and somewhat rambling genus should be absorbed into or linked with Arthrobacter we leave for taxonomists to sort out (Alderson, 1985; Goodfellow, 1987). It is, however, undoubtedly separate from Staphylococcus and is currently a valid genus. Organisms that fit the current description of micrococci are commonly encountered in routine laboratories either as environmental contaminants or as commensals from normal skin and only occasionally from infections. The difficulty is to recognize when these colonially distinct 'nonpathogens' do cause infection and, for this, simple reliable tests are required. Unfortunately, the characters that can be readily detected (Table 6.2c) are less convincing than those obtained by more complex methods such as DNA hybridization, cell-wall component analysis and the like though not all the species listed should be regarded as wholly secure. The characters shown in Table 6.2c are based on descriptions by Kloos, Tornabene & Schleifer (1974). Many of the criteria are negative yet these species can actively metabolize a wide variety of substrates; presumably the test systems are at fault in failing to detect such activity. In contrast to the staphylococci, pigment production among micrococci is a stable and important differential character; although its taxonomic significance is uncertain, we do, however, use it in Table 6.2c. The differences between M. luteus, the classical micrococcus, and M. varians are not great; they both form easily recognizable lemon-yellow, mounded colonies of varied texture. The yellow-orange colonies of M. kristinae (which behaves biochemically like a staphylococcus) and the bright orange colonies of M. nishinomiyaensis together with the pink and red colours of M. roseus and the motile M. 57

[6.2]

CHARACTERS OF GRAM-POSITIVE BACTERIA

Table 6.2c. Third-stage table for Micrococcus and Stomatococcus

Motility Oxidase VP Pigmentation Carbohydrates, acid from Glucose Fructose Sucrose Arginine Nitrate reduced Lysozyme Methicillin

1

2

3

4

5

6

7

8

9

_ + Y

_ + Y

_ + C

_ + + Y

_ + O

_ + C

+ + R

_ + R

_ — + -

— s s

+ + d + r s

— d s

+ + + r s

d + r s

+ s r

_ s s

d d + s s

+ + + + 9 S

1 Micrococcus luteus; Micrococcus afermentans; Micrococcus lysodeikticus; Staphylococcus afermentans 2 Micrococcus varians; Micrococcus lactis; Staphylococcus lactis 3 Micrococcus lylae 4 Micrococcus kristinae 5 Micrococcus nishinomiyaensis 6 Micrococcus sedentarius 7 Micrococcus agilis 8 Micrococcus roseus; Staphylococcus roseus 9 Stomatococcus mucilaginosus Y C O R

= = = =

yellow cream orange red

s = sensitive r = resistant

Other symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

agilis differ from any staphylococcal pigmentation and are usually obvious though a few isolates, particularly when young, may cause some confusion. None of the species are regarded as clinically significant though there are occasional reports of infections with strains similar to M. lylae and the marine species, M. sedentarius (Old & McNeill, 1979; Marples & Richardson, 1980; Fleurette et ai, 1987). These strains, which are of uncertain taxonomic status, are described as non-pigmented, able to hydrolyse arginine and resistant to methicillin. Stomatococcus mucilaginosus, an oral commensal organism in man, may be associated with occasional opportunist infections. It can be recognized by the sticky adherent nature of colonies on solid media and by a positive aesculin reaction together with the test characters shown in Table 6.2c. In summary, the micrococci comprise mostly environmental and saprophytic strains. The main question is one of recognition and exclusion except for the 58

occasional strain possibly associated with disease. Certainly it is essential before discarding any isolate that might be a Micrococcus to make sure it is not a Staphylococcus. And that, fortunately, is not too difficult. Minidefinition: Micrococcus. Gram-positive cocci in small or large clusters. Nonmotile; non-sporing. Aerobic. Catalasepositive; usually oxidase-positive. Do not usually produce acetoin. Attack sugars oxidatively or not at all. Type species: M. luteus; NCTC strain 2665. 6.2.3 Aerococcus (Tables 6.2a; 6.3a, b). This genus was described and named by Williams, Hirch & Cowan (1953). The cocci, which formed the ocgroup of Shaw, Stitt & Cowan (1951), were isolated from environmental samples such as air, dust, and dairy utensils, and were regarded as intermediate between staphylococci and streptococci though near-

[6.2]

THE STAPHYLOCOCCI AND MICROCOCCI er to the latter. The catalase reaction is feeble and may be described in different ways by different observers; to some it will be regarded as negative, to others it will be weakly positive. Since the catalase reaction is important for distinguishing between staphylococci and streptococci, we include the characters of Aerococcus viridans in Table 6.2a among the Gram-positive, catalase-positive cocci (Staphylococcus, Micrococcus) as well as in Tables 63a and c with the catalase-negative streptococci. Like streptococci, the aerococci do not reduce nitrate to nitrite; however, production of the enzyme leucine aminopeptidase by all streptococci (Table 63b) but not by staphylococci, micrococci or aerococci provides a basis for their separation. Minidefinition: Aerococcus. Gram-positive cocci in pairs, fours, and small clusters. Non-motile; non-sporing. Aerobic and microaerophilic; facultatively anaerobic. Catalase feebly positive or negative; oxidasenegative. Attack sugars fermentatively without gas production. Type species: A. viridans; NCTC strain 8251.

6.3

The streptococci (Streptococcus, Lactococcus, Enterococcus, Aerococcus, Leuconostoc, Pediococcus and Gemella) Characters common to members of the group: Gram-positive cocci; non-motile (rare exceptions). Aerobic, facultatively anaerobic. Catalase-negative; oxidasenegative. Carbohydrates fermented; gas not produced. Nitrates not reduced. Indole and H2S not produced.

With so many shared characteristics, this group of genera is fairly homogeneous and does not include any unexpected members except Gemella, which is somewhat different; we give the reasons for including it here in Section 6.3.6. We have also included the strictly anaerobic peptococci and peptostreptococci (see Section 6.4) in Table 63a because there can be difficulties in distinguishing them from streptococci and other members of this group. In addition to its inclusion in the staphylococcus-micrococcus group (Section 6.2.3), Aerococcus viridans appears here because it is an organism inter-

Table 6.3a. Second-stage table for Streptococcus, Enterococcus, Aerococcus, Gemella, Pediococcus, Leuconostoc, Peptococcus and Peptostreptococcus 1 Growth in air + 10% CO 2 Gas from glucose Growth in 6.5% NaCl broth Growth on Acetate Agar at pH 5.4 Growth in 40% bile Sensitive to metronidazole 1 2 3 4 5 6 7 8

+ D -

Streptococcus (pyogenic, viridans and lactic divisions): see Table 6.3b. Enterococcus (Streptococcus: enterococcus division): see Table 6.3b. Aerococcus: see Tables 6.2a; 6.3b. Gemella: see Table 6.3b. Pediococcus: see Table 6.3b. Leuconostoc: see Table 6.3b. Peptococcus: see Table 6.4. Peptostreptococcus: see Table 6.4. P. putridus; Streptococcus putridus

Symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

59

[6.3]

CHARACTERS OF GRAM-POSITIVE BACTERIA

Table 63b. Third-stage table for Streptococcus, Enterococcus, Lactococcus, Pediococcus, Aerococcus and Gemella 1 2

Haemolysis

p

Requires CO 2 for growth Growth a t 4 5 ° C Growth in 6.5% NaCl broth Growth on 40% Bile Agar Leucine aminopeptidase Bile-aesculin test Voges-Proskauer test^ Pyrrolydonylarylamidase Phosphatase Pyridoxal or cysteine dependence Hydrolysis of hippurate aesculin arginine starch c Sensitive to bacitracin (0.1 unit) optochin H 2 O 2 production Fermentation of pyruvate ribose arabinose * mannitol sorbitol adonitol sucrose lactose trehalose raffinose inulin starch Polysaccharide from sucrose Motility Yellow pigment Lancefield antigen

_

1 2 3 4 5 6

a P a b

p/-

3

4

P

a

5

6

P

p

_ + _ _ + + -

_ _ _ _ _ _ da + _ _ + + + _ _ _ + _ _ _ _ _ + + + -

_ d + _

+ + _

_

_

_

_

_

+ _ _

d _ _

d _ _

d _ _

_ _

_ _

_ _

_ + _

_

_ -

_ _

+

P

p/-

_

_

_ d

d +

_ +

.

+

_ d +

9

d

-a _ + + _ _ _ _ _ _ _ + _ _ _ _ + + + -

_ _ _ _ _ + + d _ _ _ _ _ D ^ _ _ _ d + _ _ _ _ _ + + + + + + d + + + + + _ _ _ _ _ _ _ _ _ _ + d + + + _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ A B C C C

Streptococcus pyogenes; S. haemolyticus 7 Streptococcus agalactiae Streptococcus equi 8 Streptococcus dysgalactiae Streptococcus zooepidemicus Streptococcus equisimilis 9 Streptococcus spp. group C 10 11

8

_ _

_

_ -

_

7

_

-a d +

+

_ d

10

P

a/-

_

_

_

_ _ + _ + _ + -

11

12

p/-

P

_ _

d d

d

+ _ + d

d _ -

_

-

_

d +

_ _

a/p

+

d - d + + d + _ _ _ _

-

+ _ _

_ _

_ _

d d d _ _ _ _ _ _ _

_ +

+

+ + _

-

Streptococcus spp. group G (large colony variety); Streptococcus canis Streptococcus anginosus; \S. milleri'; minute colony haemolytic streptococci; S. intermedius-MG; S.constellatus Streptococcus porcinus; 5. lentus Streptococcus uberis Streptococcus suis

Green zone around colonies on Blood Agar Clear, colourless zone around colonies on Blood Agar Some strains will grow in 4% NaCl broth More strains positive in a medium containing pyruvate than in Glucose Phosphate Broth c Zone of clearing greater than 2 mm diameter around growth on Starch Agar stained with Lugol's iodine d Strains of M-type 6 and M-type 55 ferment mannitol

_

_

_

_

d _ +

+

_

-

d _ +

_ _

_

_

_ _ _

_ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ d + + _ _ _ _ _ - - + + _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ + + + + + + + + + + d d d d d + + + + + + d + + + + + + + + + d d _ _ d d D ^ - d d d d _ _ _ _ H - d — d d — — — + + d d d + + d d d d _ _ _ _ _ _ _ _ Dx/ /Dx — — _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ C G -/F/ E/P/ -/E R/S/ L -/H -/O/KG/C/A U/V/V+

_

+ + +

+ d -

17 + _ + _ + _

_ + _

_ + d _

_

_

_

+

16

-

-

_ d

15

_ D _

+ +

+

_

+

_ + + _

+

-

d

_

+

a/p

d _

_

14

a

_

_ ~a

+

13

_ _ _ _ _ (w) (w) (w) _ _ -

12 13 14 15 16

Streptococcus spp. group L Streptococcus pneumoniae; pneumococci Streptococcus sanguis Streptococcus oralis; '5. mitior' Streptococcus spp. pyridoxal or cysteine dependent 17 Streptococcus morbillorum

e Strains of Lancefield serological Group R ferment raffinose / May be opposite reaction with strains of serotypes d or g g Positive reactions with strains of serotypes b, d and g h Usually positive with human isolates j Both antigens may be present in a single strain Dx -Dextran (glucan) Lv = Levan (fructan)

Other symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

[6.3]

THE STREPTOCOCCI

Table 6.3b. (contd).

18

19

20

21

22

Haemolysis Requires CO2 for growth

-/P a/- _ _ _

Growth a t 4 5 ° C Growth in 6.5% NaCl broth Growth on 4 0 % Bile agar Leucine aminopeptidase Bile-aesculin test Voges-Proskaiier t e s t b Pyrrolydonylarylamidase Phosphatase Pyridoxal orcysteine dependence Hydrolysis of hippurate aesculin arginine starchc Sensitive to

4 44 4 4 4 4 _ d 4+ _

d 4 + 4 - 4 - 4 4 - 4 - 4 + _ _ _ _ _

optochin H 2 O 2 production Fermentation of pyruvate ribose arabinose mannitol sorbitol adonitol sucrose lactose trehalose raffinose inulin starch Polysaccharide from sucrose Motility Yellow pigment Lancefield antigen

_ —

_ d

18 19 20 21 22 23

4+ + 4 _ + 4 4 4 _ _ _ D

Enterococcus faecalis; S.faecalis Enterococcus faecium Enterococcus mundtii Enterococcus casseliflavus Enterococcus gallinarum Enterococcus avium

-

_

+

_ +

24

25

26

a/- P a -/a/p -/a _ _ _ _ _

4 - 4 - 4 4444 - 4 - 4 4 - 4 - 4 4 - 4 - 4 4 - 4 - 4 4 - 4 _ _ _ -

_ —

23

27

a/- a _

-

4 - 4 - 4 - d d 44 - 4 - - a - a - 4 - 4 - 4 - 4 - 4 4 - 4 - 4 - 4 - 4 4 - 4 - 4 - 4 - 4 4 - 4 - 4 - 4 - 4 - 4 4 - 4 - 4 - 4 - _ _ _ _ _ -

_ —

d d 4 - 4 - 4 + _ i -

_ d

— + + 44 4 4 + + + + - d d — _ _ _ _ + + + + - 4 - 4 - 4 - 4 - 4 - 4 - 4 - 4 d d 4 - + d 4 4 - d -/w + _ _ _ _ _ _ _ + + _ + + _ D D D D

_ —

_ —

_

_ —

—

29

_ _ - a - a d d + 4d d - 4 - _ c l _

-

4_

_ —/

_ —

d 4 - / 4 # _ _ _

_ -

a P a b

29 30 31 32 33 34

31

_ - a 44d 4 d _ _

32 33

34

_ + _ 44d 44d 4d 4d - 4 - 4 - d d d _ _ _ _ _

_ 4w 4+ _

d d 4 - 4 - 4 - d + + _ _ _ _

_ d

(w) — — _ — — _ _ _ + + d 4 d d A + d + - d 4— — 4-/ — + _ _ _ _ _ _ _ + 4 - + + + 44 - 4 - 4 - 4 - 4 - 4 - 4 - d 4 - d - 4 - d + + d + + d d d d 4 d + d _ Dx/-D x Lv/Dx _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ Q/D> D D D D -IE -IK

24 Enterococcus durans; S. faecium var durans; S. durans 25 Streptococcus bovis biotype I 26 Streptococcus bovis biotype II 27 Streptococcus equinus 28 Streptococcus mutans, serotypes c, e and f

30

-/a/p - / a - / a - / a a p/a/+ __ _ _ _ _

d - a 4 44- 4 - _ -

4-

28

_ +

_ -

+ _ + d d

_

_ 4 - d d d d — _ _ _ d + + d d d d d 4 - 4 - 4 d d d + Dx/ _ _ _ _ _ _ N D/- -

_

_

d

_ d d d _ _ -

Streptococcus salivarius Lactococcus lactis Leuconostoc spp. Pediococcus spp. Aerococcus spp.; Pediococcus; Gaffkya spp. Gemella haemolysans; Neisseria haemolysans

Green zone around colonies on Blood Agar e Strains of Lancefield serological Group R ferment raffinose Clear, colourless zone around colonies on Blood Agar / May be opposite reaction with strains of serotypes d or g Some strains will grow in 4% NaCl broth g Positive reactions with strains of serotype b More strains positive in a medium containing pyruvate than in Glucose h Usually positive with human isolates Phosphate Broth j Both antigens may be present in a single strain c Zone of clearing greater than 2 mm diameter around growth on Starch Dx =Dextran (glucan) Agar stained with Lugol's iodine Lv = Levan (fructan) d Strains of M-type 6 and M-type 55 ferment mannitol Other symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

CHARACTERS OF GRAM-POSITIVE BACTERIA

mediate between the catalase-positive and catalasenegative Gram-positive cocci with characteristics of both groups; within that spectrum it is nearer to the streptococci than to the staphylococci. 6.3.1 Streptococcus (Tables 6.3a, b). Rational classification of the streptococci remained problematical until the demonstration of serologically identifiable polysaccharide group antigens by Lancefield (1933, 1940) and the definition by Sherman (1937) of four major divisions which are still in current use: the pyogenic streptococci, the viridans streptococci, the enterococci and the smallest group, the lactic (milk) streptococci. Subsequent efforts to correlate Lancefield's serological groups with divisions based on cultural and biochemical characteristics were bedevilled by a host of exceptions as well as by aberrant strains. Isolates that do not match any welldefined streptococcal species continue to occur; some, such as pyridoxal-dependent strains (Bouvet et al., 1985) might form a useful species or taxon, but others differ only slightly from species currently recognized. The application of newer taxonomic techniques, especially DNA hybridization, cell-wall analysis, and fatty acid and enzyme profiles, have certainly revealed natural relationships among, for example, the pyogenic streptococci (Kilpper-Balz & Schleifer, 1984); in contrast, the results of such tests alone would suggest that organisms as different as the oropharyngeal commensal Streptococcus salivarius and the lactic organism Streptococcus (now Lactococcus) thermophilus should form a single species (Farrow & Collins, 1984a) though the former is of no use as a starter culture for the production of yoghurt (Marshall, Cole & Phillips, 1985). The possible return of the pneumococcus to the former genus Diplococcus now seems unlikely but the idea that enterococci should form a separate genus, Enterococcus, has been revived (Schleifer & KilpperBalz, 1984; Collins et al., 1984) is now widely recognized. Despite the mass of information currently available on the characterization of streptococci, haemolysis is still of considerable differential as well as predictive pathogenic use even if limited in taxonomic value; this is reminiscent of the 1920s (Brown, 1919) when streptococci were divided into the haemolytic (those producing p haemolysis), the viridans (those produc62

[6.3]

ing green or a haemolysis) and the non-haemolytic (unfortunately mislabelled y haemolytic) on Blood Agar plates. Although the kind of haemolysis on this medium is not always clear-cut, especially as the hydrogen peroxide produced by some streptococci can interfere with haemolysin activity and contribute to 'greening' by its action on haemoglobin, it is nevertheless a useful starting point when present. In the present divisions, the pyogenic streptococci include many of the common pathogens of man and domestic animals: most but not all of them are (3 haemolytic and serological methods are particularly useful for their identification (Table 63b). Among the viridans streptococci (which include the pneumococci) and the enterococci (the latter belonging to Lancefield serological group D but having differing haemolytic properties), biochemical and physiological tests are more satisfactory than serology for species identification; the enterococci are generally more salt-tolerant, and also more heat-resistant than other streptococci. These traditional characterization procedures, despite their limitations, are usually adequate if time-consuming but they do not constitute a single comprehensive identification scheme for all the streptococci isolated in diagnostic laboratories. As a step towards such an objective, Colman & Ball (1984) suggested that miniaturized identification systems supplemented with a few other tests (including Lancefield grouping, bacitracin and optochin sensitivity and pyridoxal dependence) might suffice for the majority of strains. Outside the medical, dental and veterinary fields, the lactic streptococci, now transferred to the new genus Lactococcus (Schleifer et al., 1985), are important to the milk, dairy and cheese industries; as they are rarely if ever pathogenic, we show L. lactis only in Table 63b. The concept of 'species' as defined for streptococci is in general lower and less firm than for most other genera, it is therefore important to appreciate that 'aberrant' strains do occur and that in practice exceptions to the characters shown in Table 63b are likely to be encountered from time to time. Moreover, the precipitin reactions for the Lancefield antigens are not always straightforward; difficulties can occur, particularly with Group D strains, some extracts of which will not react with otherwise satisfactory antisera unless the antigen is precipitated with ethanol (Smith & Shattock, 1962). Some strains of S.

THE STREPTOCOCCI

sanguis and S. salivarius do not react with any of the grouping antisera and their identification depends on other characters. Many streptococci seem to be sensitive to oxygen on first isolation so that their detection in clinical material may be more successful under anaerobic conditions. Added CO2 is often advantageous for growth and some strains of pneumococci will not grow without it initially (Austrian & Collins, 1966) though the requirement is usually lost rapidly on subculture. Among the pyogenic streptococci, S. pyogenes (Lancefield Group A) remains the major pathogen of man. Usually the first step in its recognition is the presence of p haemolytic colonies on Blood Agar plates due to the oxygen-stable ' S ' lysin; many isolates, however, are only weakly haemolytic and slashing the agar during inoculation of plates or anaerobic incubation often increases the extent of haemolysis with such strains. Some isolates lack the 'S' lysin completely and are therefore non-haemolytic; the need for investigation of such strains, including determination of their Lancefield group antigen, may become apparent only because they are bacitracin-sensitive (Maxted, 1953) or because of their biochemical reactions. All Group A streptococci produce pyrrolydonylarylamidase (PYRA) but fail to ferment ribose; this combination of characteristics distinguishes them from strains of Groups B, C and G (Table 6.3Z?); it can, however, occur among other streptococci including strains of enterococci, pneumococci and aerococci as well as S. suis though not among those of S. anginosus (often called 'S. millerV by clinical microbiologists) that cross-react with group A antiserum. We suspect that in the future such biochemical tests may well replace some of the present conventional methods for the identification of the pyogenic streptococci. Strains of S. pyogenes can be typed serologically by determination of the M and T protein antigens; the latter is stable but, unlike the former, is not related to virulence. Recent changes in the pattern and severity of infections with S. pyogenes as well as the association of particular M/T types with certain clinical syndromes (Gaworzewska & Colman, 1988) suggest that this reference laboratory procedure could usefully be applied more often for epidemiological purposes. The streptococci of Lancefield Group B (S. agalactiae) isolated from human infections are often

[6.3] not only haemolytic but may also produce pigmented colonies (Tapsall, 1987). These linked characters occur much less often in strains isolated from bovine infections (Butter & de Moor, 1967). Anaerobic incubation on media containing starch is the best method of detecting the tan-coloured pigment produced by these strains (Islam, 1977). Irrespective of their haemolytic reactions on Blood Agar plates, Group B streptococci of both human and animal origin produce, with rare exceptions, a substance that will lyse sheep or ox but not human, horse, rabbit or guineapig red cells which have been exposed to a sphingomyelinase C such as the p-lysin of staphylococci or the a-toxin of Clostridium perfringens (Sterzik & Fehrenbach, 1985). This phenomenon, known as the CAMP reaction (Christie, Atkins and MunchPetersen, 1944) can occur also with some strains of S. uberis (Roguinsky, 1969) and of Listeria monocytogenes. Several different species of streptococci possess the Lancefield Group C antigen (Table 63b)\ in addition, some strains of S. anginosus ('5. millerV) crossreact with this Group antiserum. A positive Voges-Proskauer reaction and failure to ferment ribose will distinguish the latter. Subdivision of Lancefield Group C strains is based traditionally on tests for the fermentation of lactose, trehalose and sorbitol and on the presence or absence as well as the nature of haemolysis on Blood Agar media; there is, however, a need for new and more specific tests probably based on their enzyme activities. S. zooepidemicus, an animal pathogen, occasionally causes serious infections in man (Michalcu et al., 1982; Barnham et al.9 1987) but the suggestion that it should be transferred to S. equi (Farrow & Collins, 1984Z?) because of homology in DNA hybridization results fails to take account of differences in their pathogenicity and host-specificity: S. equi causes strangles in horses whereas S. zooepidemicus causes pyogenic infections in many different animals. This objection applies also to their suggestion that the human commensals and opportunist pathogens (Efstratiou, 1983) at present called S. equisimilis and the 'pyogenes-like' strains of Lancefield Group G, should be transferred to the species S. dysgalactiae. Strains of Lancefield Group G that are pathogenic for animals have been brought together as S. canis (Clark et al., 1984; Devriese et aL, 1986); these animal 63

CHARACTERS OF GRAM-POSITIVE BACTERIA

isolates all form P-galactosidase and chymotrypsin. The 'human' strains of Group G can be distinguished from S. canis because they are more 'pyogenes-like' in fermenting trehalose and in producing p-glucuronidase, streptokinase and hyaluronidase. Some strains of S. anginosus ('S. milleri') cross-react with Group C antiserum and these have the same pattern of reactions as other members of Group C except that they ferment raffinose more often (Smith & Sherman, 1938). The minute-colony-producing organisms assembled together as 'S. millerV are serologically heterogeneous and taxonomic agreement has yet to be reached; however, as the name S. milleri is still in widespread clinical use, we continue to give it alongside the present correct name S. anginosus. They are, however, biochemically homogeneous (Table 63b) and all give a positive VP reaction, form phosphatase and hydrolyse arginine but fail to ferment either ribose or sorbitol and do not produce p-glucuronidase (Ruoff & Ferraro, 1986). They can be regarded as forming one aggregated species ('£. millerV) irrespective of their haemolysis (Colman & Williams, 1972) or, alternatively, they may be subdivided into haemolytic strains called S. anginosus, and nonhaemolytic organisms labelled S. intermedius if they ferment lactose and S. constellatis if they do not (Facklam, 1984). Another approach, suggested by Coykendall, Wesbecker & Gustafson (1987), is to apply the name S. anginosus rather than 'S. milleri' to the single species but accept three bio types: biotype 1 strains are mostly haemolytic and do not ferment lactose or hydrolyse aesculin; biotype 2 strains, which may or may not be haemolytic, split aesculin and ferment lactose and sometimes starch; biotype 3 strains are like those of biotype 2 but additionally ferment either raffinose or mannitol, or both of them. We think, however, that the use of a single name for all these organisms is useful meanwhile because clinically they all produce similar suppurative diseases (Van der Auwera, 1985) although strains of biotype 3 occur also as commensals in the vagina (Ball & Parker, 1979; Poole & Wilson, 1979). The species S. porcinus, also known under differing names 'S. infrequens' and 'S. lentus\ is serologically heterogeneous and includes those streptococci, pathogenic for pigs, that belong to Lancefield serological Groups E, P, U and V. These P-haemolytic 64

[6.3]

streptococci possess a distinctive set of physiological and biochemical characters (Wessman, 1986) by which they can be identified, though full characterization would also require demonstration of their actual Group antigen (Table 63b). Some strains of S. uberis cross-react with Group E and others with Groups G, P or U antisera (Roguinsky, 1969, 1971). Useful reactions for the differentiation of these viridans streptococci from S. agalactiae include the fermentation of mannitol and sorbitol, hydrolysis of aesculin and hippurate (Cullen, 1969) and a positive VP reaction; the absence of heat, salt and bile tolerance distinguishes them from the enterococci. S. suis, another serologically heterogeneous species which is pathogenic for pigs, includes more than eight distinct serotypes. Of these, serotype 1 is known also as Group S and serotype 2 as Group R streptococci. Again, confirmation of the serotype is an aid to identification of this species, particularly in occupation-related cases of meningitis caused by Group R strains. Early descriptions of these strains suggested a greater physiological homogeneity than has been found subsequently (Hommez et al., 1986). Streptococci of Group L have been isolated from a variety of animal sources and occasionally from man. They can be mistaken for S. pyogenes (Group A streptococci) because they not only produce Phaemolysis but are often sensitive to bacitracin and may even cross-react with the 'A' reagent in some commercial grouping kits. Tests for the fermentation of ribose and the production of pyrrolydonylarylamidase (PYRA) distinguish them from S. pyogenes; and cross-reactions with group A sera do not occur with less sensitive precipitin tests. Despite all the characters shown in Table 6.3Z?, pneumococci are usually identified by their sensitivity to optochin, bile solubility or by agglutination with a polyvalent serum (OMNI-serum) though as always occasional exceptions occur. Some strains of S. oralis (also known as 'S. mitiof) are sensitive to optochin and bile-soluble strains of Enterococcus {Streptococcus) faecalis also occur; moreover, rough strains of pneumococci may not be lysed by bile (Lund, 1959). OMNI-serum contains antibodies to the choline-containing 'C-substance' present in the cellwalls of pneumococci as well as in some strains of S. oralis ('S. mitiof) and some batches of the

[6.3]

THE STREPTOCOCCI

Lancefield Group C reagent in grouping kits may agglutinate them (Lee & Wetherall, 1987). Other test characters are therefore necessary for correct identification. Non-capsulated strains of pneumococci isolated from outbreaks of conjunctivitis are more difficult; if they are both bile-soluble and optochin-sensitive there is little doubt about their identification (Shayegani et ah, 1982), but if they are not soluble in bile and not capsulated, procedures such as polyacrylamide gel electrophoresis of intracellular proteins may be needed for species identification (Pease, Douglas & Spencer, 1986). The aggregated 'species' S. sanguis is serologically heterogeneous and includes Groups H and W (Ball, 1985). Several subdivisions of this species have been proposed, including one with two subspecies corresponding to Groups 1 and 3 of Coykendall & Specht (1975). The presence of alkaline phosphatase distinguishes Coykendall & Specht's Group 1, which has this property, from Group 3, which does not. Strains that lack the glucosyltransferase enzymes responsible for the formation of glucan (dextran) from sucrose are now included with S. sanguis. A key characteristic of S. sanguis is its ability to hydrolyse arginine (Table 63b) as this character correlates with others including cell-wall composition (Price et al., 1986). Cell-wall analysis provided the clue that led eventually to the precise definition of the organism formerly called 'S. mitiof by Colman & Williams (1972) but now accepted as S. oralls (Kilpper-Balz, Wenzeg & Schleifer, 1985); since the name 'S. mitiof is still used widely we continue to give it alongside S. oralis. Facklam (1977) would allot the same organism either to the species S. mitis or to S. sanguis II, the latter containing the raffinose-fermenting strains. The presence in the cell wall of ribitol and the absence of large amounts of rhamnose were first noted by Colman & Williams (1965); subsequently, the presence of choline and an unusual mucopeptide structure called 'Lys-direct' (Kilpper-Balz, Wenzeg & Schleifer, 1985) confirmed that the cell-wall composition was unusual. Some of these strains form dextran from sucrose and for this reason were once classed with S. sanguis. The dextran-positive strains are generally more active biochemically and nearly all of them produce oc-galactosidase, (3-galactosidase and phosphatase as well as fermenting raffinose, lac-

tose and starch (Colman & Ball, 1984). Neither the dextran-formers nor the other members of the species S. oralis hydrolyse arginine or give a positive VP reaction. Some organisms that are similar in cell-wall composition to S. oralis (Roberts et al., 1979) require supplements of pyridoxal hydrochloride (10 |ig/ml) for growth in many culture media. They can be recognized most conveniently by satellitism around disks containing 20 |Hg of this substance. The production of pyrrolydonylarylamidase (PYRA) is a key character of these organisms. Bouvet and her colleagues (1985) describe three biotypes: one produces a- and (3-galactosidase and ferments trehalose, the second does not produce any of these enzymes but forms glucuronidase, and the third produces none of them. S. morbillorum grows better anaerobically than in air even with added CO2. Like one of the pyridoxaldependent biotypes, the only positive test characters may be the production of leucine aminopeptidase and pyrrolydonylarylamidase. Unlike Gemella species, phosphatase is not formed and the VP reaction is negative (Berger & Pervanidis, 1986). Among the streptococci that carry the Lancefield Group D antigen there are two disparate clusters of species (Table 63b). All the species will grow on Bile-aesculin Agar but, as this property occurs also in other species, a negative test result is more significant. One cluster fulfils Sherman's criteria for enterococci and will grow in 6.5% Salt Broth, survive heato

o

ing at 60 C for 30 minutes and will grow at 45 C. These organisms have now been transferred to the genus Enterococcus, and we deal with them in Section 6.3.2. The other cluster of Lancefield Group D species namely S. bovis with its two biotypes and S. equinus - are less tolerant of salt. S. bovis biotype I can be distinguished from S. mutans by the presence of the D antigen and by the active hydrolysis of starch. When cultured in sucrose-containing media, S. bovis biotype I usually produces a water-soluble glucan (dextran), and on Sucrose Agar it forms watery spreading colonies. The glucan formed by S. mutans is less soluble and adherent hard colonies, not unlike those of S. sanguis, develop on Sucrose Agar. Facklam (1972) observed variants, now called S. bovis biotype II, which are less active than biotype I; 65

CHARACTERS OF GRAM-POSITIVE BACTERIA

they can be distinguished from S. salivarius by the presence of the D antigen and the failure to form levan from sucrose. What were at first regarded as biotypes and serotypes (a-f) of S. mutans are now accepted by dental microbiologists as separate species. In medical laboratories, isolates of one or other of the serotypes c, e and f (not to be confused with Lancefield groups A-F) predominate (Perch, Kjems & Ravn, 1974) and for these the epithet mutans is retained (Table 63b). Strains of serotype 'a' are inhibited by bacitracin discs containing 0.1 unit and these are known as S. cricetus (Coykendall, 1977). S. rattus was formed for strains of serotype b; they hydrolyse arginine. Strains of serotypes d and g, collectively termed S. sobrinus, usually form peroxide and fail to ferment sorbitol. When cultured on Sucrose Agar, a white halo develops around the colonies (Beighton, 1985) presumably due to the production of a highly branched glucan (mutan). It is not difficult to identify typical strains of S. salivarius as they form soft, domed colonies on Sucrose Agar owing to the production of a fructan (levan) from sucrose. Some strains produce a glucan as well as a fructan and they form craggy colonies on Sucrose Agar. Clinical material occasionally contains streptococci whose classification is uncertain because they appear to share some properties of S. salivarius and S. bovis biotype II though they can be distinguished from both of those species. All three taxa give a positive Voges-Proskaiier reaction and hydrolyse aesculin but not arginine. The atypical strains can be distinguished from S. salivarius by their failure to form an extracellular polysaccharide from sucrose, and from S. bovis biotype II because they do not carry the Lancefield D antigen or give a positive result in the bile-aesculin test. S. salivarius will grow in media made selective by the addition of sodium azide (0.02%) and sucrose (5%); with such media it can be isolated from faeces. The transfer of S. lactis and its subspecies to a new genus, Lactococcus, was proposed by Schleifer et al. (1985) and this has now been accepted. These organisms are already regarded by dairy microbiologists as more like lactobacilli than streptococci. In medical laboratories strains are occasionally isolated, but the name L. lactis should not be applied, unless the Lancefield N antigen has been identified in the cul66

[6.3]

ture and other characters demonstrated, including the hydrolysis of arginine, growth in 4% but not in 6.5% Salt Broth and growth at 10 °C and 40 °C but not at 45 C. Similarly S. equinus is a precisely defined species and we do not think that failure of a viridans streptococcus to ferment lactose should be given over-riding importance. The presence of D antigen, a positive bile-aesculin test and the failure to hydrolyse arginine or to grow in 6.5% Salt Broth are additional minimal requirements for the identification of an isolate as S. equinus. Minidefinition: Streptococcus. Gram-positive cocci in pairs or chains. Non-motile. Non-sporing. Aerobic, facultatively anaerobic. Catalase- and oxidase-negative. Attack carbohydrates fermentatively. Type species: S. pyogenes; NCTC strain 8198. 6.3.2 Enterococcus (Tables 6.3a, b). This genus was originally proposed by Kalina (1970) for a group of streptococci first described by Thiercelin & Jouhard (1903) and known collectively as 'faecal streptococci'. Recent nucleic acid and taxonomic studies support the separation of these organisms from other Group D streptococci (Bridge & Sneath, 1982, 1983; Farrow et al, 1983; Kilpper-Balz et al., 1982; Schleifer & Kilpper-Balz, 1984). With increasing recognition of the generic name Enterococcus in clinical and other laboratories, its taxonomic position is now secure and we accord with this view. The genus currently contains several species listed in Table 63b. However, an alternative view which we prefer is to regard Enterococcus as comprising only two species: E. faecalis, and E. faecium with its five varieties. This situation has still to be resolved. Growth in 6.5% Salt Broth, survival after heating at 60 °C for 30 minutes and growth at both 10 °C and 45 C are all satisfactory characterization tests for differentiating Enterococcus from Streptococcus species (Table 63a). Growth at pH 9.6, often used previously for this purpose is not recommended as media at this pH are usually unstable. With the exception of E. faecalis, Collins, Farrow & Jones (1986) offer a concise review of the properties of this genus. Unlike the other species, E. faecalis ferments pyruvate (Gross, Hougton & Senterfit, 1975). The yellow pigment produced by E. casseliflavus may be demonstrated either

THE STREPTOCOCCI

by transferring some of the growth from a Nutrient Agar culture to a filter paper or, alternatively, by inspection of the packed cells after centrifugation of a broth culture. Two species, E. gallinarum and E. casseliflavus, are motile. Minidefinition: Enterococcus. Gram-positive cocci in pairs or short chains. Nonmotile (except two species). Non-sporing. Aerobic, facultatively anaerobic. Catalaseand oxidase-negative. Attack carbohydrates fermentatively. Type species: E. faecalis; NCTC strain 775. 6.3.3 Aerococcus (Tables 6.2a; 6.3a,b). The name Aerococcus viridans was introduced for Grampositive cocci that did not form chains, were either catalase-negative or only feebly positive and which were present in the air of occupied rooms (Williams, Hirch & Cowan, 1953). If the catalase reaction is recorded as negative then Aerococcus must be distinguished from the streptococci; and if positive, from staphylococci and micrococci (Section 6.2). Although aerococci closely resemble pediococci, Aerococcus is now accepted as a separate genus; however, the organism isolated from lobsters, previously called Gaffkya and subsequently Pediococcus homari (Deibel & Niven, 1960) as well as 'P. urinae-equi' (Whittenbury, 1965£; Sakaguchi & Mori, 1969) from horses are now regarded as aerococci (Kelly & Evans, 1974). Like the staphylococci and micrococci, aerococci lack leucine aminopeptidase and this differentiates them from the streptococci which possess this enzyme. In agar shake cultures, growth with nearly all strains of aerococci is maximal just below the surface and they are thus microaerophilic. Glucose is fermented with the formation of L(+) lactic acid but without gas production (Deibel & Niven, 1960). Unlike the pediococci, aerococci do not grow on Acetate Agar at pH 5.4 but they share with the enterococci (with which they may be confused) the ability to grow on 40% Bile Agar and in 6.5% NaCl Broth. On Blood Agar, colonies (<1 mm) produce zones of oc haemolysis. Aerococci have been isolated occasionally from the skin of infants, from patients with endocarditis, and from urines and wound swabs. That they can be isolated from repeated specimens taken from patients with infection suggests that they are capable of mis-

[6.3] chief, if only as opportunist pathogens (Colman, 1967; Parker & Ball, 1976). Minidefinition: Aerococcus. Gram-positive spheres in pairs, fours and small clusters. Non-motile; non-sporing. Aerobic and microaerophilic. Catalase-negative or only feebly positive; oxidase-negative. Attack sugars fermentatively without gas production. Type species: A. viridans; NCTC strain 8251. 6.3.4 Pediococcus (Tables 6.3a, b). A genus of Gram-positive, catalase-negative, lactic-acid-producing cocci which form pairs and tetrads and which occur in beer, meat-curing brines and fermenting vegetable juices. In the clinical laboratory, the species that cause spoilage of beer are unlikely to be encountered as they do not grow at temperatures above 30 C. Some of the diversity in this genus has been reduced by transferring 'Pediococcus urinae-equi' and P. homari (from lobsters) to Aerococcus (Kelly & Evans, 1974). Strains of pediococci similar to those present in silage, cheese and fermented sausages have been isolated from the human mouth (Sims, 1966), and occasional strains resistant to vancomycin (30 |Ug disc) from blood cultures (Colman & Efstratiou, 1987). Unlike streptococci and aerococci, pediococci grow on Acetic Acid or Acetate Agar at pH 5.4 (Whittenbury, 1965Z?). They grow on Blood Agar incubated in air; and in Glucose Broth they produce sufficient DL or L(+) lactic acid (Garvie, 1984) to give a final pH or 4 or less. Gas is not produced from glucose, nor are extracellular polysaccharides such as dextran or levan formed from sucrose. A few strains give a pseudo-catalase reaction on media containing haem compounds but not on Nutrient Agar containing glucose (0.05% w/v). Pediococci are distinguished from Leuconostoc species by their inability to produce slimy colonies on carbohydrate-containing media or to produce CO2 from glucose (Gibson & Abd-el-Malek, 1945). The characteristics of some species have been reviewed by Garvie (1984) and the properties of most strains isolated from clinical specimens are like those of P. pentosaceus. Some strains of this species carry the Lancefield group D antigen (Colman 1967) and also share some biochemical properties with these streptococci (Table 63b). 67

CHARACTERS OF GRAM-POSITIVE BACTERIA

Minidefinition: Pediococcus. Gram-positive cocci forming tetrads or pairs. Catalase-negative; oxidase-negative. Attack sugars fermentatively without gas production. Microaerophilic. Acid-tolerant. Type species: P. damnosus; NCTC strain 1832. 6.3.5 Leuconostoc (Tables 6.3a, b). Species of this genus are normally present in plant materials, dairy products and other fermented foodstuffs as well as in spoiled foods so that they are likely to be encountered in public health laboratories; such isolates are usually vancomycin-resistant (Orberg & Sandine, 1984). They have also been isolated occasionally from blood cultures (Colman & Ball, 1984) and, as with pediococci, some were resistant to vancomycin (Buu-Hoi, Branger & Acar, 1985). In the clinical laboratory, dextran-forming cultures of Leuconostoc mesenteroides may be confused initially with Streptococcus bovis biotype I because both yield watery colonies on Sucrose Agar and share other biochemical characteristics (Table 63b). The leuconostocs differ from other Gram-positive 'catalase negative' cocci in that they grow in air with or without added CO2. They form a natural group with the heterofermentative lactobacilli, especially Lactobacillus confusus and L. viridescens (Garvie, 1984), producing CO2 from glucose; this property can be demonstrated in shake cultures in an enriched medium such as the MRS Agar of de Man, Rogosa & Sharpe (1960) but not in inverted inner (Durham) tubes in liquid medium. Minidefinition: Leuconostoc. Gram-positive cocci in pairs or short chains. Microaerophilic. Catalase-negative. Attack sugars fermentatively and produce gas. May form dextran from sucrose. Type species: L. mesenteroides; ATCC strain 8293. 6.3.6 Gemella (Tables 6.3a, b). Gemella haemolysans was first described as a species of Neisseria by Thj0tta & B0e (1938) but it was later transferred to a new genus, Gemella, by Berger (1961) who also described it as Gram-negative. It is easily decolorized in Gram-stained smears but belongs with the Gram-positive bacteria close to the streptococci because of its cell-wall composition and DNA G+C ratio among other things (Reyn 1970; 68

[6.3]

Stackebrandt et aL, 1982). It has been suggested that G. haemolysans is synonymous with Streptococcus morbillorum (Facklam & Wilkinson, 1981) but, although the final classification of Gemella species is not yet settled, the balance of evidence favours its separation from S. morbillorum (Berger & Pervandis, 1986) and we follow this here by showing it separately in Table 63b; because it is so readily decolorized (or over-decolorized) we also show Gemella in Table 7.3 with Neisseria and Branhamella species because of the characteristics usually sought for those genera. It can be separated from them by its failure to form catalase or oxidase. The apparent similarity to S. morbillorum is probably due to the lack of reactivity of both organisms in the tests conventionally used for streptococci (Table 63b). There are, however, several distinguishing properties. Although CO2 stimulates the growth of both on agar media, G. haemolysans prefers aerobic and S. morbillorum anaerobic conditions. G. haemolysans gives a positive reaction in the Voges-Proskauer test and reduces nitrite but not nitrate (Berger & Pervanidis, 1986) whereas S. morbillorum gives none of these reactions. The haemolysis (a or |3) from which the specific epithet is derived occurs slowly on rabbit Blood Agar and on horse Blood Agar prepared with a Columbia base; it does not occur with sheep blood. Minidefinition; Gemella. Gram-positive (but easily decolorized) cocci. Aerobic, facultatively anaerobic. Catalase-negative, oxidase-negative. Attacks sugars by fermentation, producing little acid and no gas. Type species: G. haemolysans; ATCC strain 10379. 6.4

The anaerobic Gram-positive cocci {Peptococcus, Peptostreptococcus) Characters common to members of the group: Gram-positive cocci. Strictly anaerobic. Sensitive to metronidazole. Catalase-negative (except the type species Peptococcus niger).

Peptococcus and Peptostreptococcus (Tables 6.3a, 6.4) are genera of Gram-positive anaerobic cocci that can use peptones or amino acids as primary sources of energy. They are part of the normal flora of man and animals, especially of the oropharynx,

[6.4]

THE ANAEROBIC GRAM-POSITIVE COCCI

Table 6.4. Second-stage table for Peptococcus and Peptostreptococcus

Sensitive to discs containing Metronidazole (5 jig) Vancomycin (5 (xg) Liquoid (1000 ug) Carbohydrates, acid from Glucose Fructose Indole *Propionate from threonine *Iso-caproic acid from glucose

+ + -

+ + +

+ -

+ -

+ + +

+ + -

-

+

-

*For laboratories with gas chromatography facilities. 1 Peptococcus asaccharolyticus 2 Peptostreptococcus anaerobius 3 streptococci Symbols used in the table are explained in Tables 5.1 and 5.2 (p. 47).

colon and vagina; as opportunist pathogens they are often isolated, usually in mixed culture, from clinical material. Blood Agar containing neomycin and other antibiotics is selective for them (Watt & Brown, 1983); growth is usually slow (2-A days) and haemolysis does not occur (Watt & Jack, 1977). The identification of isolates is not easy, partly because the definitions of individual species are in a state of flux but mostly because it depends greatly on their enzyme activities and on analysis of the metabolic end-products by gas-liquid chromatography (Ezaki & Yabuchi, 1985). Recent taxonomic changes in the Gram-positive anaerobic cocci have left Peptococcus niger (a rare clinical isolate) as the sole species in the genus Peptococcus. All other species formerly in this genus have been transferred to Peptostreptococcus (Ezaki et a/., 1983; Holdeman-Moore, Johnson & Moore, 1986). In practice, all metronidazole-sensitive Gram-positive cocci may be confidently regarded as peptostreptococci. The arrangement of cells in Gramstained smears is not reliable for distinguishing peptostreptococci, which are supposed to form chains, from the peptococci, which should not. Confusion may sometimes occur with microaerophilic streptococci but, unlike the streptococci, peptococci and peptostreptococci are sensitive to metronidazole; they are also sensitive to vancomycin (5 |ig disc) in con-

trast to the Gram-negative anaerobic cocci (Section 7.2.3) which are resistant (Rogosa et al., 1958; Watt, Bushell & Wallace, 1984; Holdeman et al., 1986). Hofstad (1985) claims that sensitivity to novobiocin is a reliable characteristic of peptostreptococci but Watt, Wallace & Bushell (1986) dispute this. However, among strains of anaerobic cocci, Peptostreptococcus anaerobius can be distinguished readily by its sensitivity to Liquoid (polyanethol sulphonate) as shown by wide zones of inhibition (Wideman et al., 1976). We show this and other features which differentiate peptococci from peptostreptococci in Table 6.4, which includes also streptococci for comparison. Those who wish to study these genera and their species further should consult Holdeman, Cato & Moore, 1977; Holdeman-Moore, Johnson & Moore, 1986. Minidefinitions: Peptococcus. Gram-positive cocci, often in clusters. Strict anaerobes. Sensitive to metronidazole. Feeble fermentative powers: clinical isolates do not ferment glucose. Type species: P. niger; NCTC strain 11805. Peptostreptococcus. Gram-positive cocci, often in chains. Strict anaerobes. Sensitive to metronidazole. Sugars attacked fermentatively and gas may be produced. Type species: P. anaerobius; NCTC strain 11460. 6.5

The diphtheroids (Corynebacterium, Kurthia)

Listeria,

Brochothrix,

Characters common to members of the group: Gram-positive rods, not sporeforming. Aerobic, facultatively anaerobic. Catalase positive, oxidase negative. There is some similarity in morphology between all the members of this group for which the descriptive label 'diphtheroid' (derived from the specific epithet of Corynebacterium diphtheriae) is traditionally used in clinical laboratories although it has no taxonomic meaning. The grouping is convenient but artificial as Corynebacterium belongs to the high G+C mole % branch of the Gram-positive bacteria whereas the other genera have a low G+C mole % DNA content (Stackebrandt & Woese, 1981). Most members of the group grow better on media 69

[6.5]

CHARACTERS OF GRAM-POSITIVE BACTERIA

enriched with blood or serum though none require X or V factors. Haemin stimulates the growth of Corynebacterium matruchotii under anaerobic conditions but can be inhibitory with aerobic incubation. The classification of the vaginal organism first called 'Haemophilus vaginalis' and later 'Corynebacterium vaginale' has been resolved by the creation of a new genus Gardnerella though its actual taxonomic position is still uncertain; we deal with it in Section 7.10.2 but include it for comparison in Table 6.5b. Some notes on the current species of Corynebacterium are given in Section 6.5.1. It should be noted that C. bovis is atypical in that it is oxidasepositive and that the newly described skin commensal organism C. amycolatum does not contain detectable my colic acids. 6.5.1 Corynebacterium (Tables 6.5a, 6.5b) currently comprises animal pathogens and commensals together with some saprophytic organisms; the plant pathogens previously included in the genus have all been reclassified in the genera Arthrobacter, Clavibacter, Curtobacterium, or Rhodococcus (see Davis et al, 1984; Collins & Cummins, 1986). In the medical field some organisms previously placed in the genus are now excluded. 'Corynebacterium acnes9 is now classified in the genus Propionibacterium and we deal with it in Section 6.7. *Corynebacterium haemolyticum* and 'C. pyogenes" have been transferred to the genera Arcanobacterium and Actinomyces respectively and they are dealt with in Sections 6.6 and 6.7. 'Corynebacterium equV and 'C. hoagii' represent a single species and are now classified as Rhodococcus equi. On the other hand, the organisms previously forming the monospecific genus 'Bacterionema' and those of the species 'Brevibacterium ammoniagenes9 (associated with nappy rash in infants) have been reclassified in the genus Corynebacterium as C. matruchotii and C. ammoniagenes (Collins, 1982a, 1987). The organisms previously regarded as C. renale have been shown to represent three distinct species, C. renale, C. cystitidis and C. pilosum (Yanagawa & Honda, 1978). The organisms C. minutissimum, associated with erythrasma of the skin, and C. mycetoides, reported to cause tropical ulcers in man, have been reinstated in the genus Corynebacterium 70

Table 6.5a. Second-stage table for Corynebacterium, Listeria, Brochothrix and Kurthia

Growth at 37 °C Motility Catalase Carbohydrate attack VP H2S

+ + F/-a -

+ + + F + -b

— +c F + -

d + + d

1 2 3 4 a b

Corynebacterium Listeria; Listerella Brochothrix Kurthia One species positive. C. cystitidis. Some strains of L. grayi and L. murrayi produce small amounts of H2S (lead acetate paper method) c Catalase-positive at 20-25 °C; negative at 30 °C; also mediadependent Other symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

(Collins, 1982Z>; Collins & Jones, 1983). In addition, new species have been described: C. jeikeium (Jackman et al., 1987) for the multi-antibiotic resistant diphtheroids previously designated 'Group JK corynebacteria' (Riley et al., 1979; Ersgaard & Justensen, 1984) and C. amycolatum for some recent isolates from skin (Collins, Burton & Jones, 1988). The description of the genus is now based largely on chemical characters not easily determined in routine laboratories (Collins & Cummins, 1986). Consequently, it is sometimes difficult to distinguish corynebacteria from members of other coryneform genera such as Microbacterium, Dermobacter and Brevibacterium. Similarly, although more than 20 species in the genus represent distinct taxa, it is not easy to choose phenotypic characters that allow their differentiation. We show the characters likely to be of most value in identifying corynebacteria of medical importance in Table 6.5b. The work of Barksdale (1981) and Maximescu et al. (1974) among others has shown that a group of related species or varieties are able to produce diphtheria toxin when lysogenized by a suitable bacteriophage. The group includes the three cultural varieties of C. diphtheriae; C. pseudotuberculosis, which is traditionally accorded species status; and the strains previously designated 'C. ulcerans\ a taxon which

[6.5]

THE DIPHTHEROIDS

Table 6.5b. Third-stage table for Corynebacterium and Gardnerella

Motility Catalase Metachromatic granules Haemolysis Growth improved by blood/serum Carbohydrate breakdown - [F/O/-] Carbohydrates, acid from: glucose lactose maltose mannitol salicin starch sucrose trehalose xylose VP Aesculin hydrolysis Nitrate reduced Gelatin liquefaction Urease Arginine hydrolysis Pyrazinamidase Casein digestion Phosphatase

+ D D + F

+ + + F

+ + d + F

+ — + _ _ D

+ — + _ — + _ + _ + + + -

+ d + _ _ + d _ _ d d + + -

-d — — -

+ — -

1 Corynebacterium diphtheriae 2 'Corynebacterium ulcerans' 3 'Corynebacterium pseudotuberculosis; 'C. ovis'; 'C. pseudotuberculosis-ovis' \ Preisz-Nocard bacillus 4 Corynebacterium xerosis 5 Corynebacterium kutscheri; 'C.murium'; l C. pseudotuberculosis-murium' 6 Corynebacterium renale; 'C. renale' type I

7

8

9

10

11

12

F

F

F

F

? F?

+ F

13

dc 9

—

—

+

—

? 9

? +

14

15

16

17

18

lc

F

F

F

dc

-

-

d

?

-

-

-

dc ?

d -

d d -

d d -

_

9

+

-

-

?

d

d 9

_

9

1 Corynebacterium pilosum; 'C. renale' type II 8 Corynebacterium cystitidis; 'C. renale' type III 9 Corynebacterium minutissimum 10 Corynebacterium matruchotii; 'Bacterionema matruchotii' 11 Corynebacterium ammoniagenes; Brevibacterium ammoniagenes' 12 Corynebacterium striatum; 'C.flavidum'

13 Corynebacterium jeikeium; Group JK corynebacteria 14 Corynebacterium pseudodiphtheriticum; 'C. hofmannii' (and variant spellings of both epithets) 15 Corynebacterium bovis 16 Corynebacterium mycetoides 17 Corynebacterium amycolatum 18 Gardnerella vaginalis; 'C.vagihale'.

a Characteristic morphology - rods attached to filaments (whip-handles) b Scanty granules c Positive when Tween 80 added to medium d Occasional strains positive e Occasional strains negative D different results in the varieties (gravis, mitis and intermedius) of C. diphtheriae Other symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

for practical reasons is treated separately in Table 6.5b. This group of toxigenic organisms appears to differ from other members of the genus in being pyrazinamidase-negative and neuraminidase-positive (Barksdale, 1981). When diphtheria was common, smears prepared from the growth on Loeffler's serum slopes (or directly from throat swabs) and stained with methy-

lene blue were often sufficiently characteristic for experienced workers to report not only the presence of C. diphtheriae but also the identity of the variety. Typically, the gravis variety exhibits club-shaped and dumb-bell forms with scanty metachromatic granules though in the cells there are always areas stained in different shades of blue. The mitis type accords with the textbook picture of C. diphtheriae: pleomorphic 71

CHARACTERS OF GRAM-POSITIVE BACTERIA

rods with numerous metachromic granules and cells arranged in ' V and 'W' shapes. The intermedius variety, inappropriately named not from the characteristics of the organism but from the severity of disease in Leeds in the early 1930s (Anderson et al., 1933), is least like the textbook description, consisting of short rods with alternating bands of light and dark blue stain without any metachromatic granules: the so-called 'barred' appearance. Of the diphtheroid organisms found in the human throat, C. xerosis may mimic the gravis type of C. diphtheriae but after subculture it becomes more uniform in shape and, like other diphtheroids, tends to form bundles of cells: the so-called palisade arrangement. Of the animal pathogens, C. pseudotuberculosis ('C. ovis') and C. kutscheri ('C murium') are similar in morphology to the mitis variety of C. diphtheriae. The morphology of C. matruchotii - rods attached to filaments (whip handles) - is characteristic; and branching can also occur in aerobic or acidic conditions. The typical colony forms of C. diphtheriae are best seen on Chocolate-Tellurite Agar (Appendix A2.2.2). Most gravis strains produce 'daisy head' colonies; mitis strains yield colonies with a smooth surface, black in the centre but surrounded by a clear grey zone - the socalled 'poached egg' appearance; and intermedius strains produce the smallest colonies, grey in colour, with a smooth glistening surface. 'C. ulcerans' resembles C. diphtheriae var. gravis in some respects but can be differentiated by biochemical tests (Table 6.5b). Clinically, it is important not only to identify diphtheria bacilli by their cultural and morphological characters but also to establish whether or not they are toxigenic. This is usually performed with cultures in vitro by a modified Elek double diffusion precipitin method (see, for example, Jameson, 1965) instead of by animal inoculation. Corynebacteria may not grow well enough in Peptone Water sugars to show their fermentative abilities. It is preferable to use Serum sugars or a medium such as that of Hugh & Leifson (1953) with the appropriate carbohydrate substituted for glucose. Tween 80, and aeration, improve the growth of C. bovis so that it may produce acid from carbohydrates and give a positive VP reaction (Jayne-Williams & Skerman, 1966). Tween 80 also improves the growth of C. jeikeium. 72

[6.5] Mini definition: Corynebacterium. Grampositive rods which under most conditions of growth do not branch. Non-motile; nonsporing; not acid-fast. Aerobic and facultatively anaerobic. Catalase-positive; usually oxidase-negative. Attack sugars fermentatively or do not attack them. Type species: C. diphtheriae; NCTC strain 11397.

6.5.2 Listeria (Tables 6.5a, c). This organism, isolated from epidemic infection among laboratory animals, was first described by Murray, Webb & Swann, (1926) as Bacterium monocytogenes because among other characters it induced a monocyte response in their blood. The generic name, Listerella was chosen to honour Lord Lister, the surgeon, but as this had already been used in zoology and botany the genus was later renamed Listeria (Pirie, 1940). At one time it was thought to be closely related to Erysipelothrix although it differed from this genus in several respects and subsequent taxonomic studies have supported their separation (Davis et al., 1969; Stuart & Pease, 1972). Listeria monocytogenes is now known to be a fairly hardy non-sporing organism, widespread in soil and vegetation, which can cause a variety of infections referred to as listeriosis in a number of different animals, including man. In recent years there has been a marked increase in the reported incidence of listeriosis (Seeliger, 1961; Gitter, Bradley & Blampied, 1980) which it is difficult to ascribe wholly to better methods of isolation and identification. In farm animals infection is associated with poor-quality silage; in man, food, including pre-cooked and chilled meals, has also been implicated in its transmission (McLauchlin, 1987). Faecal carriage of Listeria is not uncommon, particularly among abbattoir workers and those involved in the meat trade. Following the recent transfer of the nonpathogenic species L. denitrificans to a new genus, Jonesia (Rocourt, Wehmeyer & Stackenbrandt, 1987a), Listeria now contains six species in addition to L. monocytogenes. The taxonomic position of two of them, L. grayi and L. murrayi, has been controversial (Seeliger & Jones, 1986) but recent genomic evidence confirms their place in Listeria (Rocourt, Wehmeyer & Stackebrandt, 1987Z?); neither of them is pathogenic for man. The remaining species,

THE DIPHTHEROIDS

L. innocua, L. seeligeri, L. welshimeri and L. ivanovii, are essentially genomic in derivation from L. monocytogenes (Rocourt, Schrettenbrunnner & Seeliger, 1983) and they are thus not easy to distinguish in routine laboratories. Like Brochothrix, which they closely resemble, all Listeria species survive and grow slowly at low temperatures (1-5 °C); this unusual ability is used to advantage for their selective isolation by 'incubation' in the refrigerator. This procedure is, however, slow so that selective and enrichment media such as Thallous Acetate-Nalidixic Acid Agar and Thiocyanate- or Thallous Acetate-Nalidixic Acid Broths (Kramer & Jones, 1969) should also be used. We can recommend a recently described selective medium that contains an aesculin-ferric ammonium citrate indicator system (Curtis et al, 1989) on which Listeria produces blackening; the best results are obtained by incubation at 30 °C. On solid blood-free media, Listeria colonies have such a characteristic clear, bright blue-green iridescent colour and appearance when viewed by obliquely transmitted white light that they can be recognized readily even on heavily contaminated plates. Equally characteristic is the 'tumbling' or 'head-over-heels' motility in broth cultures 'incubated' at room temperature. Another characteristic of the genus is the production of acetoin. We list these and other differential test characters for the identification of Listeria species in Table 6.5c. Most but not all strains of L. monocytogenes show P haemolysis on Blood Agar plates though the zones can be narrow and care must be taken not to mistake them for streptococci; unfortunately the type strain is non-haemolytic, atypical in the CAMP test and phenotypically identifies with L. innocua. Listeria species can easily be confused with corynebacteria, especially as they can grow on tellurite-containing media; they can also tolerate high concentrations of bile salts and grow well on MacConkey Agar. L. ivanovii, which gives wide zones of p-haemolysis, is important as a cause of abortion in sheep; it corresponds almost entirely with serovar 5 of the O and H antigenic typing scheme for L. monocytogenes of Seeliger & Hohne (1979). For epidemiological purposes, however, phage typing (Audurier et al, 1979; Rocourt et al, 1985) is preferable as it is reproducible and highly discriminatory.

[6.5] Minidefinition: Listeria. Gram-positive rods; motile, nonsporing; not acid-fast. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative; produce acetoin. Attack sugars by fermentation. Type species: L. monocytogenes; NCTC strain 10357. (Recommended typical strain, NCTC 11994). 6.5.3 Brochothrix (Tables 6.5«,c) is a genus consisting currently of two species, B. thermosphacta and B. campestris. The former is an important spoilage organism of film-wrapped, refrigerated meat; it has also been isolated from fish and other products such as milk and cottage cheese. The latter, isolated recently from soil and grass (Talon et al, 1988), is phenotypically similar to B. thermosphacta (Table 6.5c). There is no evidence that either of them are pathogenic for man but we include them because they may need to be differentiated from similar organisms, including Listeria, which occur in food products. Both species grow well between 4 and 25 °C with limited growth at 30 °C and none at 37 °C (Gardner, 1981; Talon et al, 1988). They are non-haemolytic and in young cultures (< 24h) two colony types frequently occur with such different morphology that the plates may be regarded mistakenly as contaminated. They do not show the blue-green iridescence exhibited by Listeria colonies under obliquely transmitted white light. Glucose added to the growth medium greatly increases colony size and the growth has a distinctly sour smell. They do not grow on media selective for lactobacilli such as the Acetate Agar of Rogosa, Mitchell & Wiseman (1951) and poorly, if at all, on the MRS medium of de Man, Rogosa & Sharpe (1960). They may be isolated on the selective streptomycin-thallous acetate-cyclohexamide agar (STAA) medium of Gardner (1966) on which Listeria and lactobacilli fail to grow. The organisms are catalase-positive but production of the enzyme is dependent on both the growth medium and the temperature of incubation (Davidson, Mobbs & Stubbs, 1968); Blood Agar base is usually suitable and incubation should be at 20-25 °C and not at 30 °C. Minidefinition: Brochothrix. Gram-positive rods; occur singly or in chains; non-motile; non-sporing; not acid-fast. Aerobic and facultatively anaerobic. Catalase-positive; oxi73

CHARACTERS OF GRAM-POSITIVE BACTERIA

[6.5]

Table 6.5c. Third-stage table for Listeria, Brochothrix and Kurthia

Anaerobic growth Motility P-haemolysis (horse Blood Agar) CAMP test with Staphylococcus aureus Rhodococcus equi Blue-green colony colour: oblique illumination (24 h) Bird's feather growth: Nutrient Gelatin slant Catalase Growth at: 4°C 37 °C 45 °C Growth with 8% NaCl (2 days) 10% NaCl (2 days) Carbohydrate breakdown [F/O/-] Carbohydrates, acid from: glucose mannitol a-methyl-D-mannoside L-rhamnose sucrose D-xylose VP Hydrolysis of: Aesculin Hippurate Phosphatase Nitrate reduced H2S produced 1 2 3 4 5 6 7 8 9 10 11

1

2

+ +a + b

+ -

+ -

-

+ -

+

+

— +

+

4

3

+

+ a

+

a

+

6

5

+

+ a

+

7

a

+

+ a

+

a

9

10

11

+ -

+ -

_ + -

_ + -

+c

-

-

+

-

-

-

-

-

-

+

+

+

+

+

-

-

-

-

+

+

— +

_ +

+

_ +

-

+ +

+ +

+ + w#

+ + w#

+ + w#

+ +

+ + w#

+ + w#

+ + w#

+ — —

+ _ —

+ d -

+ + +

+ + F

+ + F

+ + F

+ + F

+ d F

+ + F

+ + F

+ d F

F

d

d

_e -

_e -

+ + + — +

+ + d d +

+ — d ? + +

+ — + d ? + +

+ d + +

+ + + — +

+ + + d — +

+ + ? + + +

+ d ? + d + +

— _

— — _

+ + + -

+ + + -

+ + + -

+ + + -

+ + + -

+ +/ d

+ +/ + d

+ + -

+ + + -

-

-

+ d

+ + -

w

-

-

Listeria monocytogenes; Listere Ha monocytogenes Listeria innocua Listeria seeligeri Listeria welshimeri Listeria ivanovii Listeria grayi Listeria murrayi Brochothrix thermosphacta Brochothrix campestris Kurthia zopfii Kurthia gibsonii

a Motility must be tested at 20-25 °C b Not all strains of L. monocytogenes are p haemolytic — the type strain (ATCC 15313) is non-haemolytic c A very wide zone of p-haemolysis (or multiple zones) exhibited by L. ivanovii d Catalase positive at 20-25 °C; negative at 30 °C; also media dependent e A few strains grow with 10% (w/v) NaCl / Weak phosphatase production with some strains g About the upper limit for growth of Listena — some strains may give poor growth at 45 °C Other symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

74

8

+

d

— +

d

THE DIPHTHEROIDS

[6.5]

dase negative. Produce acetoin. Acid produced from a wide variety of sugars by fermentation. Type species: B. thermosphacta; NCTC strain 10822.

rods (actinomycetes); between these two subgroups are organisms with intermediate characters. We deal with the actinomycetes in Section 6.7 but for comparison include the genus Actinomyces in Tables 6.6a,b\ we describe it in Section 6.7.1 but for completeness include the minidefinition in Section 6.6.6. 6.5.4 Kurthia (Tables 6.5a,c) is not usually regarded as a pathogen, but we include it as strains Orla-Jensen (1919) divided the lactobacilli into have been isolated from meat and dairy products and three subgenera, lThermobacterium\ 'Streptobacterium' occasionally from clinical material (Keddie, 1981). and lBetabacterium\ on the basis of fermentative activity and temperature of growth. These names Surface colonies are rhizoid but unlikely to be have no standing in nomenclature now but it is a tribconfused with Bacillus species; they are not ute to Orla-Jensen's work that the species of the haemolytic. Two species, K. zopfii and K. gibsonii, genus Lactobacillus can still be divided into three are recognized though other psychrophilic strains similar groups of thermobacteria, streptobacteria and occur with many Kurthia-like characters (Shaw & betabacteria broadly equivalent to those of Kandler & Keddie, 1983; Keddie & Shaw, 1986). Growth occurs in the range -5 ° to 35 °C for K. zopfii and -5 ° to Weiss (1986); these groups are shown in Table 6.6a. Two of the groups are homofermentative (sugars fer45 °C or more for K. gibsonii. They give negative mented mostly to lactic acid) and the third (betabacreactions in most of the usual biochemical tests. The teria) are heterofermentative (sugars fermented partly 'bird's feather' growth on Nutrient Gelatin slopes to lactic acid and partly to volatile acids including (Keddie & Shaw, 1986) is highly distinctive for both CO2). The species characteristics of some of the lacspecies. tobacilli are given in Table 6.6b. Mini definition: Kurthia. Gram-positive rods in chains; motile but non-motile variThough normally regarded as catalase-negative, ants occur; not acid-fast. Strictly aerobic. some strains of lactobacilli (also some leuconostocs Acid not produced from sugars in peptone and pediococci) produce a catalase in media containmedia. Acetoin not produced. Nitrate not ing only low concentrations of glucose. These 'pseureduced. Type species: K. zopfii; NCTC docatalases' are insensitive to sodium azide strain 10597. (Johnston & Delwiche, 1962) and are not produced when the organisms are grown on media containing 1% glucose (Whittenbury, 1964). 6.6 The coryneform-actinomycete The genus Erysipelothrix has some resemblance to intermediate group Lactobacillus; it has also often been compared to and (Lactobacillus, Listeria, Erysipelothrix, combined with Listeria (see Section 6.5) and we Arachnia, Actinomyces, Arcanobacterium) therefore show these organisms in Table 6.6a for Characters common to members of the comparison together with Brochothrix and Kurthia. group: Gram-positive rods, usually diphWe also show Erysipelothrix rhusiopathiae in Table theroid in morphology but may show 6.7 for comparison with the propionibacteria of medirudimentary branching; non-sporing; not cal significance. acid-fast. Attack sugars fermentatively. The monospecific genus Arachnia resembles the Mostly facultative anaerobes. genus Propionibacterium in producing propionic acid Despite pressure to be more definitive, we have as the major-end product of carbohydrate retained the descriptive term 'coryneform-actinometabolism. Arcanobacterium haemolyticum (formycete intermediate' as used previously in this merly Corynebacterium haemolyticum) closely Manual for this large group of organisms because resembles the genus Actinomyces. they have relatively few common characters other Some genera and species in this large group are than 'coryneform' cell morphology. The organisms shown in more than one of the Sections and Tables. can be divided broadly into two subgroups: (i) the This arrangement is deliberate so that additional charlactic-acid producing 'bacilli' and (ii) the branching acters can be introduced and direct comparisons thus 75

CHARACTERS OF GRAM-POSITIVE BACTERIA

[6.6]

Table 6.6a. Second-stage table for Lactobacillus, Listeria, Brochothrix, Kurthia, Erysipelothrix, Arachnia, Arcanobacterium and Actinomyces 10 Oxygen requirements: Strictly aerobic Strictly anaerobic Facultatively anaerobic/microaerophilic Motility Catalase Growth at 5 °C Growth at 15 °C Gas from glucose Carbohydrates, acid from: arabinose maltose melezitose salicin VP Nitrate reduced H2S production-/ 1 2 3 4 5 6

Kurthia spp. Brochothrix spp. Listeria spp.; Listerella Erysipelothrix spp. Lactobacillus; group I, thermobacteria Lactobacillus; group II, streptobacteria

+

+

11

_b

a

w/-

-f D

D

D

D d D D D D

7 8 9 10 11

Lactobacillus; group III, betabacteria Arachnia propionica Arcanobacterium haemolyticum; 'Corynebacteriwn haemolyticwn' Actinomyces spp. Clostridium perfringens (asporogenous variants); C. welchii

e Occasional species show some growth at 5 °C a Motility must be tested at 20-25 °C / A few species grow at 15 °C b Motility (by peritrichous flagella) occurs in a few species g Strains of serovar 1 may produce acid but is usually lost on sub-culture h L. grayi and L. murrayi do not produce acid c Catalase produced when incubated at 20-25 °C; also media i L. murrayi reduces nitrates dependent d A few species may produce 'pseudocatalases' on low-glucose j H2S production in Triple Sugar Iron Agar containing media k A. viscosus is catalase positive Other symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

made between a greater number of similar or possibly similar organisms. For these reasons we show as stated the characters of Actinomyces in Table 6.6 but deal with the genus in Section 6.7.1. We would remind readers that the minidefinitions of genera, especially of those seldom seen in clinical laboratories, do not necessarily apply to all species in them as they have been drawn up essentially with those species of medical concern in mind. 6.6.1 Lactobacillus (Tables 6.6a, b) is a large genus with some 50 species. They occur in vegetation and dairy products and in man where they form part of the normal flora of the gut and vagina. Apart from their association with dental caries (Rogosa et a/., 1953) lactobacilli are generally regarded as non76

pathogenic, but there are reports of their involvement in human disease; L. casei subsp. rhamnosus as well as L. acidophilus, L. gasseri, L. plantarum and occasionally L. salivarius have been associated with bacterial endocarditis, septicaemia and other infections (Sharpe, Hill & Lapage, 1973; Berger, 1974; Bayer et al, 1978; Bourne et al., 1978; Lorenz et aL, 1982; Dickgiesser, Weiss & Fritsche, 1984). Doderlein's bacillus is not a bacteriological entity; most lactobacilli from the human vagina seem to be L. acidophilus (Rogosa & Sharpe, 1960). Some organisms isolated from meat and fish, notably L. piscicola (shown to be synonymous with L. carnis) and L. divergens, have been reclassified recently in a new genus Carnobacterium (Collins et aL, 1987) as C. piscicola and C. divergens respec-

[6.6]

THE CORYNEFORM-ACTINOMYCETE INTERMEDIATE GROUP

Table 6.6b. Third-stage table for Lactobacillus, Listeria, Erysipelothrix, Arachnia, Arcanobacterium and Actinomyces

Growth at 15 °C Growth at 45 °C Gas from glucose Carbohydrates, acid from: arabinose galactose lactose maltose mannitol melezitose melibiose raffinose salicin sorbitol trehalose Aesculin hydrolysis Nitrate reduction Arginine hydrolysis 1 2 3 4 5 6 7 a b c d

Lactobacillus acidophilus Lactobacillus jensenii Lactobacillus delbrueckii Lactobacillus salivarius Lactobacillus gasseri Lactobacillus casei Lactobacillus plantarum

1

2

3

4

5

6

7

8

9

10

11

12

13

14

_ + -

_ + -

_ + -

_ + -

_ + -

+

+ +

_ d +

+

+ -

9

-

+ -

d -

? ? -

-

— + + + d d + d + _ -

+ d d + — + + _

d d d — d — d

— + d +b +b +b — — +b +b +b + _ -

+ d d + _ + d -

d + + + -

d _

+ + _ d d _

d

+ d d — d d + d + _ -

d + + + + d + + + + + +

+

+ + + + + + d + + d _ -

-

+

+

_c —

8 9 10 11 12 13 14

_ d d + + _ + _ + + _ -

_ + + + _ — — _ _ _ +

d/ + + + + d + d d d + -

+ + d — — 7 _ d d -

D D + d D D D D D -8

D D D -

Lactobacillus brevis Lactobacillus fermentum (not 'L.fermentV; now also includes L. cellobiosus) Listeria monocytogenes; Listerella monocytogenes Erysipelothrix rhusiopathiae; E. insidiosa Arachnia propionica; Actinomyees propionicus Arcanobacterium haemolyticum; Corynebacterium haemolyticum Actinomyces spp.

Only L. casei subsp. rhamnosus grows at 45 °C L. casei subsp. tolerans does not produce acid from these carbohydrates Only those strains previously named 'L. leichmanni' hydrolyse aesculin Some strains reduce nitrate but only if glucose limited to keep pH above 6

e 45 °C about upper limit of growth for Listeria / Strains of serovar 1 may produce acid g Some strains of A. israelii and A. pyogenes may produce acid

Other symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

tively; sporing forms of lactobacilli have been classified in the genus Sporolactobacillus (Kitahara & Suzuki, 1963). None of these genera is regarded as pathogenic and they are not considered in this Manual. The species composition of Lactobacillus has changed considerably in the last decade; many new species have been described, but others have been reduced in rank. For example, L. bulgaricus and L. lactis are now regarded as subspecies of L. delbrueckii; and L. cellobiosus as a biovar of L. fermentum. Of the three groupings of lactobacilli (thermobacteria, streptobacteria and betabacteria) the heterofermentative betabacteria can produce gas from glucose under certain conditions. Such gas production is not likely to be confused with that from clostridia (and

their non-sporing variants) which is vigorous and occurs in a variety of media incubated anaerobically. In contrast, heterofermentative lactobacilli (and L. delbrueckii) produce ammonia from arginine (Kandler & Weiss, 1986). For the sugar reactions of lactobacilli, the MRS medium (Appendix A2.6.25) of de Man, Rogosa & Sharpe (1960) is usually used; Whittenbury (1963) showed that a soft agar medium was advantageous. The characters of lactobacilli likely to be isolated in medical laboratories are shown in Table 6.6b but we do not show the subspecies of L. delbrueckii, L. casei and L. plantarum. The specialized methods used in the detailed characterization of lactobacilli are outside the scope of this Manual; we refer readers for information to papers by Sharpe (1979, 1981) and Kandler & Weiss (1986). 77

CHARACTERS OF GRAM-POSITIVE BACTERIA

Minidefinition: Lactobacillus. Gram-positive rods often coryneform; chain formation common; typically non-motile and nonsporing. Not acid-fast. Microaerophilic or facultatively anaerobic. Catalase-negative. Complex growth requirements; grow best about pH 6.0. Attack sugars fermentatively. Type species: L. delbrueckii; ATCC strain 9649. 6.6.2 Listeria (Tables 6.5a, c; 6.6a, 2>) was at one time regarded as closely related to Erysipelothrix and placed in that genus. However, Barber (1939), supported later by numerical taxonomic work (Davis & Newton, 1969; Davis et al., 1969) always maintained the separation of the two genera. Stuart & Pease (1972) thought that Listeria resembled the enterococci and Erysipelothrix the other streptococci. The catalase test and motility do, however, distinguish between them. We describe Listeria more fully in Section 6.5.2 but repeat the minidefinition here. Minidefinition: Listeria. Gram-positive rods; motile; non-sporing; not acid-fast. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative; produce acetoin. Attack sugars by fermentation. Type species: L. monocy to genes; NCTC strain 10357. (Recommended typical strain NCTC 11994). 6.6.3 Erysipelothrix (Tables 6.6a, b). Until recently this genus contained only one species, E. rhusiopathiae, the causative organism of swine erysipelas and human erysipeloid. Despite agreement about its pathogenicity it has a chequered history, having been placed at various times in no less than eleven different genera with fourteen different epithets (Shuman & Wellmann, 1966). This nomenclatural difficulty was finally resolved with the issue of Opinion 32 (1970) conserving the name Erisipelothrix which is now included in the Approved Lists of Bacterial Names (1980). A second species E. tonsillarum (not 'E. tonsillae'), has been described recently on the basis of DNA hybridization but as it cannot yet be distinguished from E. rhusiopathiae by morphology or biochemical tests (Takahashi et al., 1987) we mention it only for completeness. Both species are microaerophilic and grow well on Nutrient Agar 78

[6.6]

especially with the addition of serum (5-10%); crude, but not purified, bile salts (Hutner, 1942) and Tween 80 (Ando, Moriya & Kuwahara, 1959) seem to act well as substitutes for serum. The optimum temperature for growth is 30-37 °C within the range 5—42 °C. White & Shuman (1971) showed that the fermentation reactions of Erysipelothrix strains varied with the medium and the indicator used, but most of them show a consistent pattern under the same conditions of growth. They recommended a basal medium with peptone, meat extract and 10% horse serum. We have obtained reliable results with Nutrient Broth sugars containing phenol red as the indicator, but known strains should always be included as controls (Jones, 1986). Tests for the production of H2S should be done preferably in Triple Sugar Iron Agar (Wood, 1970). Twenty-two serovars of E. rhusiopathiae have been distinguished on the basis of heat-stable somatic antigens. Serovars 1 and 2 are common; the others are rare (Kucsera, 1973; Wood, Haubrich & Harrington, 1978; N0rrung, 1979). All the strains of E. tonsillarum examined so far belong to serovar 7 (Takahashi et al., 1987). Minidefinition: Erysipelothrix. Gram-positive rods, often in filaments; not branching; non-motile; non-sporing; not acid-fast. Microaerophilic and facultatively anaerobic. Catalase negative; oxidase-negative; H2S-positive. Acid produced from a wide variety of sugars by fermentation. Type species: E. rhusiopathiae; NCTC strain 8163. 6.6 A Arachnia (Tables 6.6a, &; 6.7), a diphtheroid rod that may show filaments with branching, which can grow aerobically but prefers anaerobic conditions; CO2 is not required for either aerobic or anaerobic growth. As it is catalase-negative, Arachnia appears in the first-stage table (6.1) in the same major grouping as Lactobacillus and Erysipelothrix and hence we include it in Tables 6.6a and b. Morphologically, Arachnia resembles the actinomycetes and physiologically the propionibacteria; we therefore show its characters also in Table 6.7 and give additional information about it in Section 6.7.3. Minidefinition: Arachnia. Gram-positive diphtheroid or branched rods and filaments. Non-motile; not acid-fast. Facultatively aer-

THE CORYNEFORM-ACTINOMYCETE INTERMEDIATE GROUP

obic but grows best anaerobically. Catalasenegative. Attack sugars by fermentation with propionic and acetic acid as the main endproducts. 6.6.5 Arcanobacterium (Tables 6.6a,b; 6.7). A genus of microaerophilic, Gram-positive, irregular rods containing the single species A. haemolyticum, formerly called Corynebacterium haemolyticum (Collins, Jones & Schofield, 1982). The species was previously closely associated with Corynebacterium pyogenes (now Actinomyces pyogenes) but it has since been shown to be distinct by numerical taxonomic, chemotaxonomic and genomic techniques (Schofield & Schaal, 1981; Collins et al, 1982). The organisms were first isolated from nasopharyngeal and other infections among American soldiers in the Pacific and, as the name implies, the colonies are (3haemolytic on Blood Agar (Hermann, 1961). In young (18 h) Blood Agar cultures, Gram-positive slender rods predominate which become granular, segmented and in older cultures may appear as cocci. They grow poorly on media without the addition of blood or serum and prefer anaerobic conditions. An atmosphere with added CO2 considerably enhances growth. Serum should not be used for fermentation tests as false positive reactions may occur. The tests important for characterization are shown in Tables 6.6a and b. Minidefinition: Arcanobacterium. Grampositive, irregular rods, may appear coccoid; non-motile, non-sporing. Microaerophilic. Typically catalase-negative. Attack sugars by fermentation. Type species: A. haemolyticum; NCTC strain 8452. 6.6.6 Actinomyces (Tables 6.6a,b; 6.7). This genus is discussed in Section 6.7.1 but for comparative purposes we show the generic characters in Tables 6.6a, b and repeat the minidefinition here. Minidefinition: Actinomyces. Gram-positive rods which may show rudimentary branching. Non-motile; non-sporing; not acid-fast. Microaerophilic and anaerobic. Catalase-negative (two exceptions). Attack sugars fermentatively. Type species: A. bovis; NCTC strain 11535.

6.7

[6.6]

The actinomycetes (Actinomyces, Propionibacterium, Arachnia, Rothia and Bifidobacterium) Characters common to members of the group: Gram-positive, short to filamentous rods with branching, even if rudimentary. Non-motile; not acid-fast. Aerial mycelium not produced. Attack sugars fermentatively.

The term 'actinomycetes' is not easy to define. Some taxonomists use it to refer to Mycobacterium, Nocardia, Streptomyces and related groups; others use it to encompass all Gram-positive bacteria with a high DNA G+C content (> 55 mol %). We use it as in the previous edition of this Manual for a diverse group of organisms which show true branching but which have wide variations in atmospheric requirements between the genera and species. Moreover, they all belong to the 'actinomycete' branch of the Gram-positive bacteria as defined by Stackebrandt & Woese (1981). With the exception of Rothia, which grows best in air (Gerencser & Bowden, 1986), the organisms in this group are anaerobic or microaerophilic; with some, notably Actinomyces, added CO2 improves growth and others may also benefit. The colonial morphology of the organisms varies from smooth to rough 'breadcrumb-like' formations, both of which can be displayed by the same strain thus suggesting that the culture may be mixed; some are filamentous or spider-like. The test characters, including pigmentation, can also vary considerably with the composition of the medium and the pH as well as with the concentration of oxygen so that for species identification particular attention to media and cultural conditions is essential. Further information, with illustrations, is given in the reviews by Slack & Gerencser (1975), Schaal & Pulverer (1981), Gerencser & Bowden (1986), Cummins & Johnson (1986), Schaal (\9S6a,b) and Scardovi (1986). All the members of this diverse group ferment glucose and are sensitive to penicillin and the (3-lactam antibiotics. Their cultural characters, which are shown in Table 6.7, are not always stable and we warn against placing too much reliance on them individually; identification of this group of organisms more than most should be based on a summation of 79

CHARACTERS OF GRAM-POSITIVE BACTERIA

characters. As a rule of thumb, the more pigmented an isolate, the less likely it is to be pathogenic. 6.7.1 Actinomyces (Table 6.7). There is a wide range of morphological types amongst this genus. Most strains of Actinomyces israelii are filamentous with a rough colonial morphology, while many strains of A. naeslundii, A. viscosus and A. odontolyticus are more diphtheroidal in cell morphology with smooth colony forms. Growth of all strains is enhanced with added CO2, though Actinomyces meyeri (formerly Actinobacterium meyeri) grows best and should be cultured under anaerobic conditions (Cato et al., 1984). For growth Actinomyces require a rich medium, such as that provided by a meat infusion broth or a medium with added serum. Differentiation between species based solely on either cell or colonial morphology is not feasible. Table 6.7 shows the tests most useful for characterization; these have been chosen as reasonably reproducible, taking into account the variation likely to occur with different media. We do not include two recently described species, A. denticolans and A. howellii (the latter catalase-positive), isolated from dental plaque in cattle (Dent & Williams, 1984a, b). The common cause of actinomycosis in man is A. israelii, the pus from lesions usually containing yellowish to brown particles, the so-called 'sulphur granules'. Until recently the cervicofacial region was the site most frequently affected but during the last decade there has been a marked increase in isolations of A. israelii from women using intrauterine contraceptive devices (IUDs) or vaginal pessaries. Actinomyces species isolated from other clinical infections include: A. meyeri from brain abscesses; A. eriksonii, now regarded as a Bifidobacterium (Scardovi, 1986), from throat and abdominal lesions; and A. naeslundii and A. viscosus from the tonsils and mouth where they occur normally as commensals but may be associated together with A. odontolyticus in dental decay and with carious teeth. The classical animal infection is 'lumpy jaw' in cattle caused by the type species, A. bovis. Other species associated with animal infections of veterinary concern include A. pyo genes (formerly Corynebacterium pyogenes) causing mastitis in cattle (Reddy, Cornell & Fraga, 1982); A. israelii, causing infections similar to A. bovis; A. suis, which affects 80

[6.7]

the soft tissues of cattle; A. viscosus from the teeth of hamsters; and the recently isolated A. hordeovulneris, a cause of canine actinomycosis (Buchanan et al., 1984). The increasing incidence of A. israelii infections as well as recent research into dental decay have led to improvements in the isolation and characterization of these microorganisms which were previously regarded as 'unusual'. The most reliable identification techniques are those based on classical tests (Slack & Gerencser, 1975; Schofield & Schaal, 1981) rather than on commercial rapid test kits, which have not yet proved reliable for these organisms. Although characters are given in Table 6.7 for differentiation of the more common species, the classification of the genus is still far from satisfactory. However, there appear to be three distinct groupings consisting of: (1) A. israelii; (2) A. naeslundii, A, viscosus and A. odontolyticus; and (3) A. bovis and A. pyogenes. A. israelii is a well-defined species which can be readily distinguished from others (Holmberg & Nord, 1975; Pine, 1970; Schofield & Schaal, 1981; Schaal, 1986a); it possibly deserves recognition as a separate genus within the Actinomycetaceae. A. bovis, although the type species of the genus, differs from all the other species in its cell-wall composition (Schleifer & Kandler, 1972; Schaal, 1986a) and in its biochemical characteristics (Schofield & Schaal, 1981). The only species with which it can be associated is the recently described A. pyogenes, previously classified as Corynebacterium pyogenes (Collins & Jones, 1982; Reddy, Cornell & Jones, 1982; Reddy, Cornell & Fraga, 1982). There may thus be some justification for removing A. bovis from the genus though this would create nomenclatural difficulties and we would prefer the status quo to be maintained meanwhile. A. viscosus produces catalase, will grow aerobically and does not hydrolyse aesculin or reduce nitrite. Minidefinition: Actinomyces. Non-motile Gram-positive rods which may show true though rudimentary branching. Nonsporing; not acid-fast. Microaerophilic and anaerobic. Catalase-negative (two exceptions). Attack sugars fermentatively. Type species: A. bovis; NCTC strain 11535. 6.7.2 Propionibacterium (Table 6.7). A genus of heterogeneous Gram-positive bacteria that produce

[6.7]

THE ACTINOMYCETES

Table 6.7. Second-stage table for Propionibacterium, Arachnia, Actinomyces, Rothia, Bifidobacterium and Erysipelothrix 10 Growth in air Growth anaerobically CO2 required for growth CO2 improves growth Catalase Carbohydrates, acid from: glycerol lactose maltose mannitol raffinose salicin sorbitol sucrose trehalose xylose melezitose melibiose VP Aesculin hydrolysis Starch hydrolysis Nitrate reduced Indole Gelatin liquefaction Growth in 10% bile

w/-

w/-

1 Propionibacterium acnes; Corynebacterium acnes; Voss' C. acnes', group I; C. parvum (Cummins & Johnson, 1974). 2 Propionibacterium granulosum; Voss' C. acnes group II 3 Propionibacterium avidum 4 Arachnia propionica; Actinomyces propionicus 5 Actinomyces bovis 6 Actinomyces pyogenes; Corynebacterium pyogenes 7 Actinomyces israelii 8 Actinomyces naeslundii a b c d e

W /_

C

£0

dC

11 w/-

12

14

13

15

c

_d -d -d

(w) d

D D (W)

D D D 9

(d)

9 9 9 9 9

9 Actinomyces odontolyticus 10 Actinomyces viscosus; Odontomyces viscosus; the hamster organism 11 Actinomyces meyeri 12 'Actinomyces eriksonii'; probably Bifidobacterium spp. (Scardovi, 1986)) 13 Rothia dentocariosa; Nocardia dentocariosa 14 Bifidobacterium spp. Lactobacillus bifidus 15 Erysipelothrix rhusiopathiae; E. insidiosa

Catalase reaction improved if culture exposed to air before testing Better growth anaerobically Poor growth may occur in air; growth improved with CO2 but best anaerobically Some growth anaerobically with added CO2; Schofield & Schaal (1981) report stimulation of growth by CO 2 Some strains of Bifidobacterium bifidus are negative

Other symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

propionic acid as one of the end-products of their fermentative metabolism. It contains two main groups of organisms with different habitats: those from cheese and dairy products; and those which occur as commensals in the skin and the respiratory and intestinal tracts of man and animals. The former, which represent the original members of the genus and are thus often referred to as 'classical' propionibacteria (see Cummins & Johnson, 1986), are not

considered here. The latter, exemplified by the anaerobic acne bacillus, occur frequently as contaminants of clinical material and as part of a mixed microbial flora in opportunist infections. The acne bacillus, originally ill-placed in the genus Corynebacterium, was eventually transferred to Propionibacterium as proposed by Douglas & Gunter (1946) but only after a protracted debate, until agreement early in the 1970s, about whether or not 'C. acnes' was represen81

CHARACTERS OF GRAM-POSITIVE BACTERIA

tative of all the anaerobic diphtheroids. Clinically, P. acnes and P. granulosum are the two species most frequently isolated from purulent infections, though previously there was some doubt about the separate identity of the latter species (Voss, 1970; Johnson & Cummins, 1972); unlike the other representatives of the genus neither of them will grow in air. With freshly isolated strains, variation in characterization test results does occur, particularly with aesculin and casein hydrolysis and in the carbohydrate fermentation patterns; however, some reports of such variation in results between different laboratories may have been due to mixed cultures (Cummins & Johnson, 1986). The taxonomic status of the anaerobic species P. lymphophylum, which has been isolated from urinary tract infections, is doubtful; it may well not be a member of the genus (Cummins & Johnson, 1986). Minidefinition: Propionibacterium. Grampositive diphtheroidal rods; may be coccoid, bifid or even branched. Non-motile; nonsporing. Anaerobic to aerotolerant. Catalase-positive. Attack sugars by fermentation with propionic acid as the main end product. Type species: P. freudenreichii; NCTC strain 10470. 6.7.3 Arachnia (Table 6.7). As its name implies, one important character differentiating this single species genus, Arachnia propionica, from Actinomyces is the production of propionic acid from the fermentative breakdown of glucose, now usually detected by gas chromatography. Diaminopimelic acid is also present in the cell wall (Johnson & Cummins, 1972). Originally described as an actinomycete (Buchanan & Pine, 1984), this organism is facultatively aerobic but grows best under anaerobic conditions; CO2 is not required (Schaal, 1986Z?) and the finding of Schofield & Schaal (1981) that it may be stimulatory under certain cultural conditions remains to be clarified. Arachnia propionica can cause clinically typical actinomycosis, and it may be difficult to distinguish from cultures of Actinomyces israelii contaminated with Propionibacterium acnes. Isolation techniques to ensure purity are thus, as always, of paramount importance. The colonies of A. propionica are rough and the cells are usually filamentous and may be 82

[6.7]

branched. It can be distinguished from Actinomyces israelii not only by analysis of the end-products of fermentation but also by its inability to ferment melibiose, melezitose and xylose or to hydrolyse aesculin (Table 6.7). The two serovars recognized within the species differ from each other in some of their physiological characteristics (Schaal, 1986Z?). Minidefinition: Arachnia. Gram-positive diphtheroidal or branched rods and filaments. Non-motile; not acid-fast. Facultatively aerobic but grows best anaerobically. Catalase-negative. Attack sugars by fermentation with propionic and acetic acids as the main end-products. Type species: A. propionica; ATCC strain 14157. 6.1 A Rothia (Table 6.7). This group of catalasepositive Gram-positive rods, represented by the single species R. dentocariosa, was previously contained within Nocardia until Georg & Brown (1967) created a new genus for it. Unlike Nocardia, it ferments carbohydrates and never produces conidia or a mycelium. R. dentocariosa has been isolated from carious teeth but its role in infection remains unclear. The organism usually grows best under microaerophilic conditions and not at all under anaerobic conditions. It produces rough, wrinkled colonies on solid media; microscopically it shows branching filaments which fragment easily and appear diphtheroidal or even coccoid as the culture ages; fresh isolates fragment less easily. Strains grow in the presence of 10% bile and produce ammonia from serine. For further information, see Gerencser & Bowden (1986). Minidefinition: Rothia. Gram-positive diphtheroidal or filamentous rods; pleomorphic but do not form a mycelium or produce aerial hyphae. Non-motile; not acid-fast. Microaerophilic and aerobic. Catalase-positive. Attack sugars by fermentation. Reduce nitrite to ammonia. Type species: R. dentocariosa; NCTC strain 10917. 6.7.5 Bifidobacterium (Table 6.7) species are mainly diphtheroidal in morphology but are characterized by crudely branching forms, often T or Y shaped. The majority of strains require anaerobic growth conditions. Bifidobacteria occur in the human oral cavity, intestinal tract and vagina and in the fae-

THE ACTINOMYCETES

ces and rumen contents of animals and birds (Reuter, 1971; Scardovi et al., 1971; Slack & Gerencser, 1975; Scardovi, 1981). The taxonomic status of the genus is uncertain, as are several of the 20 species given in the Approved Lists of Bacterial Names (1980). A variety of fermentation patterns have been described and this is the usual method for speciation. We make no attempt at species characterization and show in Table 6.7 only their collective characteristics. Bifidobacterium bifidum, previously known as Lactobacillus bifidus, is probably the best known species and, because of its limited ability to attack sugars, is the easiest to identify (Scardovi et aL, 1971; Scardovi, 1981, 1986). They resemble lactobacilli in cell-wall structure and carbohydrate metabolism (Kandler, 1970) but we believe current taxonomic work is likely to show that Bifidobacterium is separate from both the lactobacilli and the actinomycetes. Minidefinition: Bifidobacterium. Grampositive rods with a tendency to form rudimentary branches. Non-motile; non-sporing; not acid-fast. Anaerobic. Catalase-negative. Attack sugars by fermentation. Type species: B. bifidum; ATCC strain 29521. 6.8

The anaerobic bacilli (Clostridium, Eubacterium) Characters common to members of the group: Gram-positive rods; spore-forming (except Eubacterium); not acid-fast. Anaerobic. Sensitive to metronidazole.

The degree of anaerobiosis required by members of this group ranges from the strict {Clostridium tetani, C. chauvoei, Eubacterium species) through the less exacting species (C perfringens) to the facultative aerobe C. histolyticum. The techniques for the culture of anaerobes have become more sophisticated and greater use is now made of commercially available anaerobic cabinets for all manipulative procedures, although anaerobic jars still play a major and satisfactory role in most general laboratories. In the past, many species were undoubtedly created too readily among anaerobic organisms, sometimes on the basis of a single isolate or character. However, following promulgation of the Bacteriological Code and the Approved Lists of Bacterial Names, several

[6.7] species with previously well-known names (e.g. ' C tetanomorphum* and 'C. subterminale') have been discarded either because viable cultures with the characters listed were not found extant or because they were shown to be identical with other approved species. The species of current medical interest, which now include for example C. barati and C. ramosum are shown in Table 6.8. C. putrefaciens, despite its name, is not strongly proteolytic and is unlikely to be isolated often as it grows best at room temperature but not at 37 °C; we include it for comparison as it is fairly salt-tolerant and is associated with the spoilage of brine-cured ham and other foods. We also continue to include Eubacterium, a genus of Gram-positive bacilli which do not produce spores, in Table 6.8 to obviate the remote possibility of confusion with non-sporing variants of clostridia. 6.8.1 Clostridium (Tables 6.6a, 6.8). In this genus, species characterization in the diagnostic laboratory depends partly on spore morphology as well as on tests for particular enzyme activities. For those with gas-liquid chromatography facilities, identification can be assisted by the detection of other metabolic products. Many species of clostridia can be further subdivided into varieties or types by the different toxins (enzymes) they produce (Sterne & Warrack, 1964). However, as few bacteriologists have the specific antitoxins available or the expertise to carry out the tests, we do not give third-stage tables to show these toxin types; such isolates should be referred if necessary to a reference laboratory. Those interested in the finer subdivisions are referred to publications by Brooks, Sterne & Warrack (1957) for Clostridium perfringens; Oakley, Warrack & Clarke (1947) and Oakley & Warrack (1959) for C. novyi\ and Smith (1977) for C. botulinum. It should be noted that C. sporogenes and proteolytic strains of C. botulinum are closely similar genetically and in their general cultural characters, the only significant difference between them being the production of a neurotoxin by C. botulinum although the former is usually haemolytic. The clostridia of wound infections are dealt with extensively by Willis (1969, 1977, 1988). Infections caused by clostridia, including sporadic cases of enteritis, are not generally transmissible from person to person, so that in outbreaks of foodpoisoning due to certain toxin types of 83

[6.8]

CHARACTERS OF GRAM-POSITIVE BACTERIA

Table 6.8. Second-stage table for Clostridium 1 2 Motility Spore position and shape Growth at37°C Carbohydrates, acid from: glucose lactose sucrose salicin Nitrates reduced Indole Gelatinase Caseinase Urease Egg yolk: lecithinaseC lipase

3

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20

21

22

23

+ - - + + + + - + + + + + + + + + + + + + UX UX UX TX VX VX TXY TX VX VX VY UX UX VX VX VX VX VX VX TY VX + + + + + + + + + + + + + + + + + + + + +

D VX +

+ + + + + + + + + + + + + + + + + da + + _ _ _ _ _ _ _ + _ _ _ _

_ _

1 Clostridium butyricum 2 Clostridium perfringens; Welchia perfringens 'C. welchii 3 Clostridium barati; C. paraperfringens 4 Clostridium tertium 5 Clostridium fallax 6 Clostridium carnis 7 Clostridium ramosum 8 Clostridium innocuum

+ + + + _ _

+ - - - _ _ _ _ _ 9 10 11 12 13 14 15 16 17

+ + + + -

+ + + + + + + + + + - + + - d a d a - d a d a + _ _ _ + _ + _ +

+ d a _ +

+ + + _ _ _ d a - - - - - _ _ + +

_ _ _ _ _ _ _ _ _ _ + +

Clostridium chauvoei; C.fesseri Clostridium septicum Clostridium sphenoides Clostridium botulinum (A,B,F)* Clostridium botulinum (C,D,E)t Clostridium novyi; C. oedematiens Clostridium bifermentans Clostridium sordelli; C. bifermentans Clostridium sporogenes

* Mainly proteolytic t Mainly non-proteolytic a More strains positive than negative

T U V X Y

+

+

+ + + - _ _ _ _ _ - - - - - - - - - - - - + _ _ _ + + + ( j a + +

+ + + _ _ _ _ + _ _ + _ _ _ 18 19 20 21 22 23

_ _ +

_

_

D D D D D D D

- D

Clostridium difficile Clostridium subterminale Clostridium tetani; Plectridium tetani Clostridium histolyticum Clostridium putrefaciens Eubacterium spp.

spores terminal spores central spores subterminal/central; variable in position spores oval (ellipsoidal) spores round

Other symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

C. perfringens, and of botulism, secondary cases do not occur. In the more recently recognized association of C. difficile with pseudomembranous colitis and antibiotic-associated diarrhoea there is some evidence that hospital cross-infection may occur (Mulligan, 1988); however, epizootics due to a variety of clostridial infections can cause considerable losses among flocks and herds of animals. The isolation of clostridia is not usually difficult but their purification requires patience and skill, and may take several weeks. Isolation techniques usually involve enrichment culture of material heated at 80 °C for 10 minutes to destroy vegetative organisms; treatment with ethanol ('alcohol shock') prior to enrichment serves the same purpose. However, it is prudent to culture unheated material as well in case there are insufficient spores present in the heated 84

material to yield any subsequent growth. After incubation for 24-48 hours, subcultures are made to other media (e.g. Blood Agar plates). For C. tetani, we recommend the use of a modified Fildes' method in which the material is inoculated onto a small area of a Blood Agar plate. After incubation for about 10 hours, subcultures are made from the advancing edge of the fine growth swarming across the plate; C. tetani swarms more rapidly than other bacteria so that the edge of growth often yields the organism in pure culture. Infections and cultures may, however, be mixed with more than one clostridial (or other anaerobic) species present; their separation and identification, which may need the use of specific antisera and sometimes animal inoculation, is one of the more satisfying achievements in clinical bacteriology. Some comments on the tests for the characters

THE ANAEROBIC BACILLI

shown in Table 6.8 are needed. In Gram-stained films of the clostridia, some species, especially the strict anaerobes such as C. tetani and C. novyi, sometimes stain Gram-negatively, particularly in old cultures. The spores show as circular or oval unstained areas within the cells but they can be stained by the malachite green method. Clostridial spores may be situated centrally, terminally or in between, and they usually distend the bacillary body. Since their position is often variable (and the judgement subjective), the spore data in Table 6.8 should be used cautiously and 'flexibly'. Terminal spherical (drumstick) spores are characteristic of C. tetani, while the virtual absence of spores is characteristic of many strains of C. perfring ens and of C. fallax, which is now accepted as a species of Clostridium (Willis, 1960). Because C. perfringens does not produce spores readily detectable in cultures (including paradoxically strains isolated from material heated to destroy all vegetative organisms) and because it does not need strict anaerobic conditions for growth, we have included it also in Table 6.6a for other Gram-positive bacteria which grow better anaerobically than they do aerobically. The non-sporing eubacteria might possibly be confused with 'asporogenous' variants of clostridia though only if microscopy alone is considered. Among the common clostridia only C. perfringens and C. butyricum form capsules; all are motile except C. perfringens, C. barati and C. innocuum. The clostridia typically produce colonies of irregular shape. Some species such as C. sporogenes form frankly rhizoidal colonies; others, such as C. tetani, rarely produce discrete colonies except in young cultures, their growth characteristically appearing as a thin transparent 'swarming' film. C. perfringens is one of the few commonly encountered clostridia which forms a circular, entire colony. Haemolysis, considered alone, is not a definitive feature of clostridia. Though many species are haemolytic on Blood Agar plates, different strains of a single species may vary considerably. Some species produce more than one haemolysin, and different haemolysins vary in their activity with the nature of the red cells used. Gelatinase activity is common among clostridia. All strongly proteolytic clostridia digest gelatin, but there are many gelatinolytic species that do not attack complex proteins such as casein. For the detection of

[6.8] gelatinase activity we prefer Frazier's (1926) acid mercuric chloride method (Appendix C3.23) though it is safer to use 30% trichloracetic acid instead of the mercuric chloride solution. For proteinase activity Milk or Casein Agar is usually used; the strongly proteolytic clostridia, such as C. histolyticum and C. bifermentans, can hydrolyse a variety of complex proteins. As a useful rule, strongly proteolytic species rarely ferment lactose. The fermentation reactions of most clostridia may be determined in Peptone Water sugar media; for exacting species we recommend the plate method of Phillips (1976) (Appendix C3.8.1) The usual fermentable sugars are glucose, lactose and sucrose, with salicin as a useful addition. Strongly saccharolytic species such as C. perfringens and C. butyricum ferment all the three basic sugars; weakly saccharolytic organisms ferment only glucose. Species that do not ferment glucose do not attack any of the basic sugars and they constitute the group of 'non-saccharolytic clostridia'. Indole production is a feature only of C. bifermentans, C. sordelli, C. sphenoides and C. tetani among the species under consideration. Egg-yolk reactions, of great importance for the identification of clostridial species, depend on lecithinase C and lipase activity, both types of enzyme producing quite distinct opacity changes in Egg-Yolk Agar (Willis, 1962). Lecithinase C hydrolyses lecithin into phosphorylcholine and an insoluble diglyceride, the reaction resulting in intense diffuse opaque haloes in the medium around colonies and zones of growth; these reactions are inhibited by appropriate antitoxic sera. The lecithinase C-producing species comprise C. perfringens, C. bifermentans, C. sordelli, C. novyi and C. barati. It should be noted, however, that occasional strains of C. perfringens may fail to produce lecithinase. The lipase hydrolyses free fats into glycerol and free fatty acids, and the reactions in Egg-Yolk Agar appear as zones of intense opacity usually limited in extent to that part of the medium immediately beneath colonies or zones of growth; in addition there is a 'pearly' surface layer overlying the growth. C. sporogenes, C. botulinum and C. novyi are the lipolytic species. It is worth noting that C. novyi is the only species that produces both a lecithinase and a lipase. 85

CHARACTERS OF GRAM-POSITIVE BACTERIA

Urease activity serves to distinguish C. sordelli from C. bifermentans; indeed it is a sound practice to check the urease activity of all lecithinase C-positive isolates. The soluble 'exotoxins' produced during growth and acting on specific substrates such as erythrocytes, proteins, lecithin and lipids are responsible for many of the differential cultural characters of clostridia. Other important clostridial toxins do not have recognizable in vitro activity, and require demonstration of their characteristic pharmacological effects in vivo in animals; such toxins include tetanospasmin and botulinum neurotoxin. The enterotoxin of C. difficile also falls into this category, but is exceptional in having useful identifiable cytopathic effects on cultures of tissue cell monolayers (Donta, 1988; Levett, 1988). These effects and their inhibition by antitoxin are also used to identify the presence of the enterotoxin in faecal preparations from cases of pseudomembranous colitis. Minidefinition: Clostridium. Rods, Grampositive in young cultures; typically motile (non-motile forms occur). Not acid-fast. Produce spores that are usually heat-resistant. Anaerobic; some species facultatively aerobic. Metronidazole-sensitive. Catalasenegative. Some species attack sugars fermentatively, others not at all. Type species: C. butyricum; NCTC strain 7423. 6.8.2 Eubacterium (Tables 6.7; 6.8). These nonsporing anaerobic rods are probably quite unimportant in clinical bacteriology; they are part of the normal intestinal flora and have not been convincingly incriminated as pathogens. Holdeman, Cato & Moore, (1977) characterized 19 species, but we include only one, E. lentum, in Table 6.8 for the inexperienced in case of confusion with non-sporing variants of clostridia. Minidefinition: Eubacterium. Gram-positive rods; do not branch. Non-sporing; not acid-fast. Anaerobic. Metronidazole-sensitive. Attack sugars fermentatively or not at all. Type species: E. limosum; ATCC strain 8486. 6.9

The aerobic spore-formers (Bacillus) Characters common to members of the group: Gram-positive rods. Not acid-fast. 86

[6.8]

Grow under aerobic conditions and produce heat-resistant spores. The genus Bacillus embraces a large number of rodshaped spore-forming species with a great diversity of properties. Neither the genus nor many of the species contained within it are taxonomically homogeneous and this is reflected in the variability of many of the characters listed in the first-stage (primary) identification table for Gram-positive bacteria (Table 6.1). Apart from non-sporing variants, the variable characters include (i) the Gram-reaction, (ii) motility, (iii) ability to grow under anaerobic conditions, (iv) the oxidase reaction, and (v) the method by which carbohydrates are broken down. Of these, the variability of the Gram-reaction is the most disturbing to the neatness of the identification scheme used in this Manual because it is based on the primary division of bacteria into Gram-positive and Gram-negative organisms. Bacillus is generally regarded as a genus of Gram-positive organisms but we must emphasize that strains of some species are Gram-positive only in the early stages of growth, and others may actually be Gram-negative (Gordon, Haynes & Pang, 1973; Claus & Berkeley, 1986) even though they all have cell walls which are structurally Gram-positive in type. We continue, however, to justify the more rigid but less scientific treatment of Bacillus as a genus of mostly Gram-positive bacilli on the grounds that the important species of medical interest, particularly B. anthracis and B. cereus, are clearly Gram-positive. We would remind readers, however, that the minidefinitions in this Manual do not necessarily apply to every species in a genus. The laboratory identification of B. anthracis is a matter of some clinical urgency; the identification of other species is rarely so pressing. It should be noted, however, that B. anthracis is not the only species of medical and veterinary interest. B. cereus is now well recognized as a cause of food-poisoning (Kramer et al., 1982) and of occasional tissue infections in man and other animals (Gilbert et al., 1981; Norris et al., 1981). Several other species including B. macerans, B. sphaericus, and B. subtilis, have also been implicated in various infections, mostly in immunodeficient or severely compromised patients (Logan, 1988). It is therefore important to recognize their potential pathogenicity and to examine further specimens rather than just dismiss Bacillus isolates

THE AEROBIC SPORE-FORMERS

from clinical material as contaminants. In spite of the different reactions of Bacillus species (and of strains) shown in Table 6.1 for firststage separation, these tests do not provide an adequate basis for subdividing the genus into species. Several unsuccessful attempts have been made to subdivide it with characters such as motility, optimal temperature for growth, nutritional requirements (Proom & Knight, 1955) and chromatographic techniques (Jayne-Williams & Cheeseman, 1960) but none have given a better arrangement than that described in the monograph of Smith, Gordon & Clark (1952) based on traditional biochemical tests, spore position and morphology. This classification, which was revised by Gordon, Haynes & Pang (1973) and divides the genus into species in three groups, has been adhered to substantially ever since. In such a heterogeneous and complex genus, this is marked tribute to the excellence of their work. 6.9.1 Bacillus (Tables 6.9a,b,c). Primary allocation to this genus is made on the basis of aerobic growth of spore-forming rods. As there are now more than 60 valid species, we include in Table 6.9a only those species recognized by Smith, Gordon & Clark (1952) as well as others which can grow on ordinary media under the conditions normally used in medical laboratories. We have omitted numerous species which would not normally be isolated in a medical laboratory either because they require special medium supplements for growth or because the physical conditions such as temperature needed for growth are quite different from those of medically important bacteria. Recently, the identification of Bacillus species has been approached by methods such as pyrolysis mass spectrometry (Shute et al., 1984), pyrolysis gas chromatography (O'Donnell et al, 1980), DNA homology (Seki et ah, 1978; Krych, Johnson & Yousten, 1980; Priest, 1981) and enzyme electrophoresis (Baptist, Mandel & Gherna, 1978) though few of them have been adopted in routine diagnostic laboratories. In general the Bacillus species identified by these methods accord with those of Gordon, Haynes & Pang (1973) based on spore morphology and biochemical reactions. The latter tests are described in detail by Claus & Berkeley (1986). They are carried out at 30 °C except for the thermophile, B. stearothermophilus, which is used for checking the

[6.9] efficiency of hospital sterilizers. It will not grow at this low temperature and should be incubated at 45-60 °C. Wolf & Barker (1968) and Walker & Wolf (1971) recognized three major varieties (or groups of organisms) within B. stearothermophilus which we show in Table 6.9a. Of these, Group I includes for example 'B. thermodenitrificans\ Group II B. pallidus and Group III resembles most closely B. stearothermophilus sensu stricto; they recommended incubation at 55 °C for characterization tests with tese organisms. We have excluded the psychrotrophic species (Larkin of Stokes, 1967) which occur commonly in frozen and refrigerated foods but which grow poorly, if at all, at or above 30 °C. The shape, position and size of spores can be determined by phase-contrast microscopy, or alternatively with a spore stain such as that described by Schaeffer & Fulton (1933) (Appendix B2.3.1). Acid production within 7 days should be examined in an agar medium with an ammonium salt as the primary nitrogen source and supplemented with a little yeast extract (Appendix A2.5.5). The ability to grow anaerobically can be a useful diagnostic feature but it may be difficult to determine. Indeed several species, notably B. subtilis, described as strict aerobes can grow, albeit weakly, under anaerobic conditions; in addition, other species, such as B. licheniformis (which was originally described as an anaerobe) may lose their capacity for fermentation. Several media, broth or agar-based, have been used for this test. They include Glucose Broth and the 'anaerobic agar' of Gordon, Haynes & Pang (1973) as well as the latter medium with the additoin of 1% glucose (Claus & Berkeley, 1986). We prefer to use agar media, incubated anaerobically, as the results are usually clearer than with liquid media. Nutrient (tryptose) Agar (or even Blood Agar) may also be used in deep tubes (or plates) with or without a pH indicator provided the medium is thoroughly pre-reduced preferably for 2-3 days before inoculation. Visible growth or colour change within 7 days confirms the ability to grow anaerobically. Whatever medium is used, it is important to ensure anaerobiosis, to use appropriate control cultures and to interpret the results carefully. With the recent description of numerous new members, the genus Bacillus has become unwieldy though many of the species can still be identified by conventional tests. It can, however, be laborious 87

Table 6.9a. Second-stage table for Bacillus species

Gram reaction Chains of cells Motility* Cell length > 3jam Spore position and shape Swelling of cell body by spore Growth at 50 °C frrnwth in 10% Naf^i VJIL/YVlil 111 1VJ fO l^dv^l

Anaerobic growth Carbohydrates, acid from ASS: glucose cellobiose galactose mannose melibiose raffinose salicin xylose ONPG Utilization of citrate Urease T

A

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

+ +

+

+ +

+ +

d d

+ d

+

+ +

+ d

+ d

+ d

d d

d d

d d

_

d d

+ -

_

+

_ -

d -

+ +

d d

d -

d d

+ VX VX d _ d +

VX d

d VX -

_ VX d

VX

-

-

+ + d + d + + + +

+ + +

+

-

i

+ + + VX VX VX A •+id U + + -

d _ _ + d d

d d

-

d d _ d

d _ -

d d + d _ d

+ -

d -

+ +

+ + +

+ + +

-

+ d

+ d

+ + + + -

_ d _ d d d

d

d + + + + + -

_ d + + -

+ + + -

+ d

_ + d d d d d VX VX VTX TYX VX VX VTX VX VX VX + + + d + + + + d + + + + + + — + — + + + + + +

+ d + d + + d + +

+ + + d d + + + + d

+ d d d + + d d d

d d + + + d d d d

_ d — d d -

+ d d d + d d d

_ d d -

+ + + + + + + + -

+ + + d + -

d + + + -

+ + + + -

d d + + + -

d d + d

+ + + +

_ d + + -

d d + -

d d _ d -

+ + + + + + + + -

+ + + + + + + + d -

d + d +

+ + + + -

+ TY +

_ _ _ + VTX; vx VX VTX + d + d + + +

-

-

d

-

-

_ -

_

d + d d d d d -

d d _ d + + _ _

d + + + d -

d + + + -

+

d d d + + -

_ d d

-

1

inooie VP Nitrate reduction Casein hydrolysis Hippurate hydrolysis Starch hydrolysis Oxidase 1 2 3 4 5 6 7 8

1

Bacillus anthracis Bacillus cereus; B. anthracoides Bacillus mycoides Bacillus thuringiensis Bacillus firmus Bacillus lentus Bacillus megaterium Bacillus pumilus

+ + + + d

+ + + + d 9 10 11 12 13 14 15 16

+ + +

Bacillus subtilis Bacillus licheniformis Bacillus amyloliquefaciens Bacillus coagulans Bacillus pantothenticus Bacillus alvei Bacillus brevis Bacillus circulans

* All motile species may produce non-motik; variants T spore terminal V spore central/subterminal X spore oval (ellipsoidal) Y spore round Other symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

17 18 19 20 21 22 23 24

+

+ + d d

_ d + +

_ d d d

+ -

Bacillus laterosporus Bacillus macerans Bacillus polymyxa Bacillus sphaericus Bacillus badius Bacillus stearothermophilus (Group I: Wolf & Barker, 1968; Walker & Wolf, 1 971). Bacillus stearothermophilus (Group II: Wolf & Barker, 1968; Walker & Wolf, 1971). Bacillus stearothermophilus (Group III: Wolf & Barker , 19681; Walker & Wolf,, 1971).

[6.9]

THE AEROBIC SPORE-FORMERS

Table 6.9b. Supplementary tests for the identification ofB. anthracis and closely related species

Motility Rhizoid colony formation Crystalline inclusions Capsule formation a 'String of pearls' test b Lysis by Y phage c Haemolysis on 5% Blood Agar 1 2 3 4

+ + + -

+ +

+ +

+ + +

Bacillus anthracis Bacillus cereus; B. anthracoides Bacillus mycoides Bacillus thuringiensis

a Capsule formation after growth on Bicarbonate Agar with Serum plates incubated with 5-10% carbon dioxide, or in an anaerobic jar without catalyst, containing a carbon dioxide generator, at 37 °C overnight. Use Giemsa stain and examine microscopically for capsule formation. (Appendix A2.6.5). b 'String of pearls' formation after growth in medium containing lOu/ml Penicillin G. (Appendix C3.57). Data from Parry, Turnbull & Gibson (1983). c Data from Brown et al (1958). Symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

Table 6.9c. Supplementary tests for the identification ofB. subtilis and closely related species 1 Acid from inulin Arginine dihydrolase a-D-Arabinosidase production a-D-Glucosidase production (3-D-Glucosidase production L-Tryptophan-aminopeptidase production Anaerobic growth Propionate utilization Chain formation DNA hydrolysis CMC hydrolysis a

+ d + + d + +

d + + + d + + + d + +

— + + -

1 Bacillus subtilis 2 Bacillus licheniformis 3 Bacillus amyloliquefaciens a

Carboxymethylcellulose hydrolysis. Culture on Nutrient Agar with 0.5% CMC for 48 h at 30-37 °C. Flood plate with2M HC1 for 10 min, then Ethanol until a white precipitate is formed (about 8 h).

Symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

and computer-assisted schemes have been developed to aid identification. One scheme, claimed to give rapid and reproducible results (Logan & Berkeley, 1981, 1984; Berkeley, 1984), utilizes the API miniaturized biochemical system with 12 tests from the API20E and all of those in the API50CHB kit, together with supplementary morphological tests. An alternative probablistic scheme, described by Priest & Alexander (1988), is based on traditional tests for 30 morphological and physiological characters. It permits identification of 44 common Bacillus species, including many environmental isolates, within a few days. In the second-stage identification table (6.9a), we have used data from both of these studies. There are a few areas where species differentiation is complicated or unclear which need comment. B. cereus, B. anthracis, B. mycoides and B. thuringiensis are all closely related species which have been the subject of much controversy. B. anthracis was regarded by Gordon, Haynes & Pang (1973) as a pathogenic variety of B. cereus; they suggested also that B. mycoides and the insect pathogen, B. thuringiensis, should be regarded as sub-species of B. cereus. This is because they differ from B. cereus in only a few characteristics which are virtually all plasmid-associated and can be lost, whereupon they become indistinguishable from B. cereus by the usual characterization tests. Had Smith, Gordon & Clark (1952) followed the principles of the Bacteriological Code at that time, B. anthracis would have been the species and B. cereus the variety as the epithet 'anthracis' antedated that of 'cereus". However, they gave good reasons for regarding B. cereus as the parent form and thus for disregarding the less scientific requirements of the Bacteriological Code. In contrast, Leise et al. (1959) and Burdon & Wende (1960), citing several distinguishing features, argued that B. anthracis should remain as a separate species with its medically meaningful name. Currently, each of the four similar organisms in this group still retain species status and can be differentiated by the tests listed in Table 6.9b. It should be noted, however, that Logan et al. (1985) were able to distinguish virulent, avirulent and intermediate strains of B. anthracis from strains of each of the other species in the group. Differentiation between B. subtilis and B. licheniformis is normally achieved by virtue of the ability of the latter to grow anaerobically but it should be 89

[6.10]

CHARACTERS OF GRAM-POSITIVE BACTERIA

remembered that B. subtilis has a limited capacity to grow in the absence of air and this can be confusing. The industrially important, amylase-producing species, B. amyloliquefaciens, described by Priest et al. (1987) is easily confused with B. subtilis but can be distinguished by the tests shown in Table 6.9c. Although the species B. firmus and B. lentus are readily distinguished (Table 6.9a), several other poorly described taxa have properties similar to them (Gordon, Hyde & Moore, 1977) and are difficult to identify. Such taxa, however, are usually associated with saline or slightly alkaline environments and are less likely to be encountered in the clinical laboratory. Nevertheless, should difficulty be experienced in identifying these or other Bacillus isolates, the detailed and more extensive computer-based systems of Logan & Berkeley (1984) or Priest & Alexander (1988) should be consulted. Minidefinition: Bacillus. Rods, mainly Gram-positive in young cultures. Motile (some non-motile forms occur). Not acidfast; produce heat-resistant spores under aerobic conditions. Aerobic; some species facultatively anaerobic. Oxidase-variable; catalase-positive. Species differ in the manner in>which they attack sugars; some do not attack them. Type species: B. subtilis; NCTC strain 3610. 6.10 The acid-fast rods (Mycobacterium, Nocardia) Characters common to members of the group: Gram-positive rods, occasionally coccoid; non-motile. Acid-fast. Aerobic to microaerophilic. Attack sugars oxidatively. The characteristic acid-fastness varies in general from weak to strong from Nocardia to Mycobacterium, although this also depends to some extent on the age of the culture. Rapidly growing mycobacteria, such as M. fortuitum, certainly lose their acid-fastness in older cultures, but in practice there is rarely any difficulty in deciding whether or not a strain is acid-fast. If such a problem should arise, then smears of the strain should be decolorized, one with 25% and another with 5% sulphuric acid for 1 minute. This will help to distinguish between the genuine acid-fast strains, which are resistant to the 5% acid, and other strains which are decolorized by it. 90

Table 6.10a. Second-stage table for Nocardia and Actinomadura madurae 1 Acid fast Aerial hyphae Carbohydrates, acid from: adonitol arabinose inositol maltose mannitol rhamnose xylose Decomposition of: casein hypoxanthine tyrosine urea xanthine 1 2 3 4

d +

d +

d +

+

-

+

+

d -

+ -

+ -

+ + d d + + +

+ -

+ + +

+ + + + -

+ + -

-

d

-

Nocardia asteroides Nocardia otitidis-caviarum; N. caviae Nocardia brasiliensis; N. mexicana; N. pretoriana Actinomadura madurae; N. madurae

Symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

6.10.1 Nocardia (Table 6.10a). Although mainly soil organisms, nocardias are capable of causing infections and are isolated occasionally in medical laboratories. Since the previous edition of this Manual, opinion has consolidated and several species that were once regarded as nocardias have now been assigned to other genera. Thus Nocardia madurae, the cause of 'Madura foot', is now classified in a new genus as Actinomadura madurae (Goodfellow & Lechevalier, 1986); however, as we do not refer to this species elsewhere in this Manual, we have included it in Table 6.10a. Similarly, 'Mycobacterium rhodocrous" is now assigned to the 'plant' genus Rhodococcus (Goodfellow, 1986) but as it has no medical significance we do not include it. The definition of the genus Nocardia now rests largely on the presence, within the cells, of characteristic lipids such as mycolic acids with 46 to 60 carbon atoms in the chains (Minnikin & O'Donnell, 1983) and of large amounts of meso-diaminopimelic acid in the cell wall (Goodfellow & Cross, 1983). Tests for these acids are not, however, suitable for routine diagnostic laboratories so that, if necessary,

[6.10]

THE ACID-FAST RODS

strains should be referred to a reference laboratory. The species pathogenic for man are N. asteroides, N. brasiliensis and ' N. caviae , now renamed N. otitidis-caviarum. They can be isolated from clinical specimens by the use of Sabouraud's Dextrose Agar, Beef-heart-infusion Blood Agar, or Lowenstein-Jensen medium (Schaal, 1984). Their colonies vary in both morphology and colour depending on the composition and consistency of the medium. They usually have an aerial mycelium which makes them look 'velvety' but if this is absent the colonies are matt in appearance. The pigmentation may diffuse into the medium and often ranges from creamy-white and salmon-pink to an orange-tan colour. There are several other species of Nocardia which can be differenti-

ated by means of some 150 characters (Orchard & Goodfellow, 1980) but as they are not clinically significant we have excluded them. In general, the nocardias are rather slow-growing organisms and precise identification can take months rather than days but as the infections with which they may be associated are usually chronic, this delay is not of much consequence. Minidefinition: Nocardia. Gram-positive or Gram-variable rods and filaments which sometimes show branching. Some strains weakly or partly acid-fast. Non-motile. Produce aerial hyphae. Aerobic. Catalasepositive. Attack sugars by oxidation. Type species: N. asteroides; NCTC strain 11293.

Table 6.10b. Second-stage table for Mycobacterium

Growth at 25 °C 37 °C 45 °C Growth in presence of: PNBA thiacetazone Atmospheric preference Pigment in light Pigment in dark Tween hydrolysed

1

2

_ +

_ +

+/G +/g

A d

M -

4

5

6

+/G +/g

+/G +/g

+/ g -

+/g +/G

+/g +/G

-

+ + 9

+ +

+ +

+ + M -

+ -

9

+ + A -

+ + A + +

3

1 Mycobacterium tuberculosis*; human type of tubercle bacillus 2 Mycobacterium bovis*; bovine type of tubercle bacillus 3 Mycobacterium marinum*; M. balnei 4 Scotochromogenic psychrophils 5 Mycobacterium chelonei; M. borstelense; M chelonei 6 Mycobacterium ulcerans* 7 Mycobacterium kansasii** * ** A M V G g

7

9

+ +

8

9

9 +/g +/G

+ M + + +

10

11

12

13

+/g +/G

+/P +/£

+/g +/g

+/G +/G

+ + A d

+ + M +

+ + M d d -

+ + A _ d +

8 9 10 11 12

Flavescens group Gordonae group; M. aquae Fortuitum group; M. minettii; M. perigrinum Terrae group Avium-intracellulare - scrofulaceum group**; M. avium; M. intracellulare; M. marianum 13 Smegmatis-phlei group 14 Mycobacterium xenopi**

Always clinically significant Frequently clinically significant Aerobic Microaerophilic Variable in different growth conditions Good growth Growth but not vigorous

Other symbols used in the table are explained in Tables 5.1 and 5.2 on p. 47.

91

14 _

+ 9

V V ?

CHARACTERS OF GRAM-POSITIVE BACTERIA

6.10.2 Mycobacterium (Table 6.10b). The taxonomy of mycobacteria has benefited greatly from the studies of the International Working Group on Mycobacterial Taxonomy, an ad hoc group of bacteriologists who have examined a large number of strains by a variety of methods or by clearly defined techniques. As a result some 41 valid species of Mycobacterium were described by Kubica (1978), of which M. tuberculosis and M. bovis are well known as human and animal pathogens. Although the latter is naturally resistant to pyrazinamide, these two pathogens are probably variants of one species, Mycobacterium tuberculosis, and should therefore be termed M. tuberculosis subsp. hominis and M. tuberculosis subsp. bovis. However, as there are differences in both epidemiology and treatment, it is important to distringuish between them. For this reason they are usually regarded as separate 'species' and we accord with this traditional usage. In previous editions of this Manual, we relied on several biochemical and cultural tests for the identification of strains. In this edition, we use the scheme devised by Marks (1976) as it relies on a small number of simple tests to assign mycobacteria to species or to groups without the need to use laboratory animals. The quantitative catalase test described in the previous edition of this Manual is difficult to perform accurately and is best done in reference laboratories where numerous strains are examined as a routine. Oxidative attack on sugars also has a degree of variability when used by routine laboratories because of the long-term incubation period needed and the greater likelihood of contamination. The essential point of the scheme is that strains should be identified only as far as is clinically necessary. Further identification is possible, for example, by phage typing, agglutination tests or by thin-layer chromatography of the cell-surface lipids, but as these tests are the province of reference laboratories, they are outside the scope of this Manual. In tissues, mycobacteria often occur alone at first though secondary infection with organisms of lesser pathogenicity is liable to occur subsequently. The isolation of slowly growing mycobacteria from sputum and other material containing organisms able to grow rapidly therefore requires measures to kill or at least inhibit the multiplication of such flora. We therefore utilize the greater resistance of mycobacte92

[6.10]

ria to chemical agents and treat the material with either acid or alkali for 10 to 30 minutes. After neutralization, this material is inoculated on media suitable for the growth of mycobacteria and incubated for up to 8 weeks before being discarded as negative. For primary isolation, egg-based Lowenstein-Jensen medium containing malachite green is usually used; the same medium with the addition of pyruvate instead of glycerol is also used routinely for the isolation of M. bovis as well as for some drug-resistant strains of M. tuberculosis. As the medium must not be allowed to dry out during this prolonged period of incubation, the use of screw-capped containers is recommended. As this Manual is concerned primarily with bacteria of medical importance, the species M. paratuberculosis (Johne's bacillus), M. microti (the vole bacillus) and M. lepraemurium (the marine leprosy bacillus) have been excluded. M. leprae, the human leprosy organism, has still not been grown on artificial media and can be detected only by Ziehl-Neelsen staining of specimens or by inoculation and propagation in the nine-banded armadillo (Kirchheimer & Storrs, 1971) or the footpad of the mouse (Shepard, 1960). Growth in both of these animals is very slow and their use is for research rather than diagnosis. For growth of M. paratuberculosis, special media supplemented with mycobactin, a mycobacterial extract with an iron-chelating ability, or with killed acid-fast bacteria, are needed. Many of the slow-growing species are causally associated with chronic infections but the faster-growing mycobacteria are rarely significant. Some species such as M. tuberculosis are always clinically significant whereas others such as M. kansasii may be present as incidental contaminants. Multiple isolation of such strains from a patient, especially in large numbers, indicates clinical significance as does isolation from an aspirate or from tissues. For the identification of mycobacteria, the acid-fast nature of strains must first be demonstrated; in tissues, the organisms usually stain well but care must be taken, especially when the numbers are small, to ensure that false positive results are not reported because of the presence of contaminating acid-fast organisms in the reagents (see, for example, Carson et al., 1964). Technically, a distinction used to be made between organisms which were both acid- and

THE ACID-FAST RODS

alcohol-fast and those that were only acid-fast, but this distinction was difficult to substantiate in practice and is impracticable. Most laboratories now use a mixture of acid and alcohol for decolorization so that the organisms should strictly be described as 'acidand alcohol-fast', though the term 'AFB' for acid-fast bacilli is universally recognized and accepted. For differentiation of mycobacteria, the simple scheme shown in Table 6.10fr relies initially on the ability of strains to grow at one or more of the temperatures 25 °C, 37 °C and 45 °C. Those which grow only at 37 °C are strict mesophiles; those which grow better at 25 °C than at 37 °C are psychrophiles; those which grow better at 37 °C than at 25 °C are mesophiles; those which grow at all three temperatures are wide-range mycobacteria; and those that grow at 45 °C but not at 25 °C are thermophiles. With strict mesophiles, absence of growth in the presence of/7-nitrobenzoic acid (PNBA) and thiacetazone indicates that the strains are tubercle bacilli. Further differentiation of strains depends on oxygen preference, pigment production and the ability of strains to hydrolyse Tween 80. Where a particular test is not necessary for the identification of a species or group of mycobacteria, the result has been omitted from Table 6.10b. M. marinum has a narrower temperature range for growth when first isolated but after subculture it may grow at 37 °C. M. tuberculosis may be differentiated from M. bovis by its sensitivity to pyrazinamide, niacin production, nitrate reduction and resistance to thiophene carboxylic acid hydrazide (TCH), but these tests are usually best carried out in reference laboratories. They are also used to identify the BCG (Bacillus Calmette-Guerin) vaccine strain derived from M.

[6.10] bovis (Jenkins et al, 1985). For epidemiological purposes, M. tuberculosis may be subdivided into a number of geographical variants such as the 'Asian' and the 'African I and II' strains (Collins, Yates & Grange, 1982); the latter, isolated respectively in West and East Africa, are also called (illegitimately) 'M. africanum . All these variants differ in minor respects from classical M. tuberculosis in the tests mentioned above though defining such strains is primarily of epidemiological interest. Two geographical variants of M. chelonae also occur which differ in their ability to grow in the presence of 5% NaCl and to utilize citrate for growth (Stanford et aU 1972). Some strains of Mycobacterium produce pigment both in light and in the dark and are termed scotochromogenic, as opposed to strains which produce pigment only in the light and which are designated photochromogenic. Scotochromogenic psychrophils and the Flavescens, Gordonae, Terrae, Fortuitum and Smegmatis-phlei groups are so rarely significant in clinical practice that they do not require further speciation though M. phlei is unusual in its ability to grow at 52 °C. If clinical significance is suspected, the strain should be submitted to a reference laboratory with as much information as possible, including the number of isolations, the site and nature of the lesion and medical history. Minidefinition: Mycobacterium. Gram-positive rods that do not show branching. Typically acid-fast. Non-motile. Nonsporing and do not produce aerial hyphae. Aerobic or microaerophilic. Attack sugars by oxidation. Type species: M. tuberculosis; NCTC strain 27294.

93

Characters of Gram-negative bacteria

Although not ideal for making a major subdivision of bacteria, Gram's method of staining is convenient because everyone uses it (in one of its many modifications) when characterizing a bacterium. There is an unfortunate tendency to omit this step, especially when dealing with cultures isolated on selective media which, by virtue of the inhibitory agents they contain, may in theory affect the bacterial staining reactions; it is simply assumed that colonies on selective media have the appropriate (or expected) tinctorial and shapely qualities. This may often be tolerated (and sometimes acceptable) in busy routine laboratories where, in the words of W.S. Gilbert, it may be regarded as 'merely corroborative detail, intended to give artistic verisimilitude to an otherwise bald and unconvincing narrative'. However, our objectives are neither speed nor artistry; we want to know the identity of the organisms and for this we need accurate characterizations. We cannot afford therefore to omit making smears and staining them by Gram's method, even if we do not pursue microscopy any further. 7.1

Division into major groups

Before dealing with the Gram-negative bacteria proper, we would remind readers that there are a few organisms that seem to be on the borderline between the Gram-positive and the Gram-negative, for example Gemella, a genus which until recently was thought to consist of Gram-negative cocci. Other bacteria show an unusual phenomenon in that they develop some Gram-positivity as cultures age, in contrast to the Gram-positive bacteria, which usually become Gram-negative as the cells age and degenerate. Mobiluncus is a recently recognized genus (Spiegel & Roberts, 1984) in the former category and we suspect it will find a larger place in this Manual in 94

the future; organisms of this group, whose association with the female genital tract is well recognized, are the subject of current taxonomic interest. There is also a small number of miscellaneous Gram-negative bacteria which cannot reasonably be allocated to any of the other groupings of organisms we recognize in Table 7.1; these we have collected together in Section 7.10 as a 'Group of Difficult Organisms'. 7.2

The Gram-negative anaerobes (Bacteroides, Fusobacterium, Veillonella, Acidaminococcus, Megasphaera) Characters common to members of the group: Gram-negative bacteria; do not form spores. Strictly anaerobic. Sensitive to metronidazole.

In contrast to the previous edition of this Manual, the Gram-negative and strictly anaerobic cocci now include three genera, Veillonella, Acidaminococcus and Megasphaera, though for practical purposes the last two can be ignored; and the Gram-negative anaerobic non-spore-forming rods are represented by two genera, Bacteroides and Fusobacterium. Previously, the classification of these anaerobic rods was based mainly though not exclusively on two main criteria (1) morphological: in Fusobacterium (formerly called Fusiformis) the ends of the rods were pointed or fusiform whereas if rounded they were classified as Bacteroides', and (2) fermentation products: Fusobacterium produced butyric acid but Bacteroides did not do so. As may be imagined, the use of such weak characters for differentiation caused much confusion as well as nomenclatural difficulties (see Holdeman & Moore, 1972; Willis, 1964). In addition to poor and/or conflicting descriptions of

[7.2]

GRAM-NEGATIVE ANAEROBES

Table 7.1. First-stage table for Gram-negative bacteria 1 Shape Motility Growth in air Growth anaerobically Catalase Oxidase Glucose (acid) Carbohydrates [F/O/-]

R S

2

3 S

d D + _ 7 + D + F/- - O

4

5

6

7

8

9

10 11 12 13 14 15 16 17 18

S S / R R R R R R R R R

+ + + _ - + - O/-

- + + + + + _ + - - - F - - -

+ + + + + + + + + O O/-F

+ + F

R + + +

+ + + F

19

R R/S R WC WC D - - + + + + - * - t + D + + + + + D - D + - D + +/w + + D _ _ _ F N T - -

20 21 22 23 H R + - + + + F

+

R NT + NT + + - + + ? D ? _ ? + F - ?

Bacteroides Fusobacterium a Veillonella b Neisseria Branhamella Acinetobacter Kingella Moraxella Brucella Bordetella pertussis Bordetella parapertussis Bordetella bronchiseptica Alcaligenes Shewanella Pseudomonas (alkali-producers) Achromobacter Agrobacterium c Janthinobacterium Pseudomonas (oxidizers) d Flavobacterium e Actinobacillus Pasteurella Aeromonas salmonicida Cardiobacterium Chromobacterium Vibrio Plesiomonas Aeromonas

Enterobacteria/

Haemophilus Gardnerella Eikenella Campylobacter Helicobacter Arcobacter Anaerobiospirillum Streptobacillus # Legionella Mycoplasma * Generally poor growth in air; better growth in air + CO2. t No growth in air or anaerobically; growth in 3-10% O2. a Also Leptotrichia buccalis b Also Acidaminococcus and Megaspnaera c Also Ochrobactrum d Also Weeksella; do not attack carbohydrates e Also Chryseomonas and Flavimonas f Enterobacteria: Buttiauxella, Cedecea, Citrobacter, Edwardsiella, Enterobacter, Erwinia, Escherichia, Hafhia, Klebsiella, Kluyvera, Morganella, Proteus, Providencia, Salmonella, Serratia, Shigella, Tatumella, Yersinia and others. 8 Also Shigella dysenteriae 1: (Shiga's bacillus). Other symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

""I Cultural characters of these | organisms can be found in tables _ j with the number indicated. NT

Not testable by usual methods. Fermentative (Sneath & Johnson, 1973).

\ ^ | Typical form ? Not known

[7.2]

CHARACTERS OF GRAM-NEGATIVE BACTERIA

Table 7.2a. Second-stage table for Bacteroides 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

Growth in air + CO2 Indole Gelatinase Carbohydrates, acid from: arabinose glucose rhamnose salicin sucrose trehalose 1 2 3 4 5 6 7 8 9

18

+ -

-

db

-

-

+

+

-

+

+

+

+

+

+

+ +

+

+

d^ + + +

_ + -

+ + + +

+

dc d^ +

+ +

_ _

dc + +

db

_ +

+

-

+

+

_

_

+

+

+

_ _

_ _ +

_ _ -

+

_ + +

Bacteroides distasonis Bacteroides fragilis Bacteroides ovatus Bacteroides thetaiotaomicron Bacteroides uniformis Bacteroides vulgatus Bacteroides splanchnicus Bacteroides ruminicola" Bacteroides capillosus

10 11 12 13 14 15 16 17 18

_

_

_

+

+

+

_

+

_

+ _ _

+

_

+ _ _

—

_

d +

_

-

c

+ +

-

:

_

+ _

+

_

?

-

_ -

_

-d

_ -

-

?

?

-

?

_ —

Bacteroides melaninogenicus Bacteroides disiens Bacteroides bivius Bacteroides intermedius** Bacteroides oralis Bacteroides asaccharolyticus Bacteroides ureolyticus*; Bacteroides corrodens Eikenella corrodens Veillonella spp.

Species 1-6 = Bacteroides fragilis 'group' Species 10-14 = Bacteroides melaninogenicus - Bacteroides oralis 'group' * Urease +ve, nitrate +ve ** Some strains are lipolytic a Two subspecies recognized b More strains positive than negative c More strains negative than positive d May be positive on some media Other symbols used in the Table are explained in Tables 5.1 and 5.2 on p.47.

these organisms, their apparent sensitivity to aerobic conditions during manipulative procedures also caused considerable problems. All these difficulties led to the development of anaerobic cabinets, especially for reference work. However, Bacteroides and Fusobacterium will usually tolerate the transient aerobic conditions during routine work at the bench especially if added CO2 is used for culture (Watt & Jack, 1977). For these reasons Cowan, in the previous edition of this Manual, pragmatically refused to distinguish between these genera, recognizing only Bacteroides. Since then the separation of Bacteroides and Fusobacterium, based largely on gas-liquid chromatography of metabolic end-products, has been accepted as have several species which are now included in the Approved Lists of Bacterial Names (1980) for each genus. We show these species with 96

their differential characters in Tables 12a and b, though precise identification may take some time and their differentiation in routine diagnostic laboratories is still difficult. For practical purposes, allocation to the overall group 'Gram-negative anaerobic bacteria' is usually adequate as all of them, including Veillonella and the related genera Acidaminococcus and Megasphaera, are sensitive to metronidazole and most are catalase-negative. 7.2.1 Bacteroides (Table 7.2a). We show in Table 1.2a the characters of the species currently recognized in this heterogeneous group as well as those of Eikenella corrodens (previously included in Bacteroides) which may seem to be anaerobic on first isolation (Eiken, 1958) though it will grow in air with added CO2 on subculture (Jackson & Goodman,

GRAM-NEGATIVE ANAEROBES

1972). For comparison we also show Veillonella and related species of anaerobic Gram-negative cocci collectively in this Table. The catalase test (which we do not show) ought ideally to be negative for Bacteroides species but B. fragilis strains usually give positive reactions and other strains vary. The genus contains four clusters of species: the saccharolytic, non-pigment-forming B. fragilis group; the saccharolytic pigment-producing B. melaninogenicusB. oralis group; the saccharolytic rumen group; and B. asaccharolyticus and related organisms, some of which also produce pigment. Most clinical isolates belong either to the B. fragilis group (species 1-7) or to the B. melaninogenicus-B. oralis group (species 10-14), together with B. asaccharolyticus. Shah & Collins (1989, 1990) have proposed that the genus Bacteroides should be restricted to the B. fragilis group, and that the B. melaninogenicus-B. oralis group should be transferred to a new genus, Prevotella. The taxonomic position of many of the other species currently placed in Bacteroides is doubtful. Of the B. fragilis group, two species, B. fragilis and B. thetaiotaomicron, are commonly found as pathogens. As a rule, members of this group are resistant to aminoglycosides, penicillin, colistin and vancomycin. Surface colonies on horse Blood Agar are circular, entire, convex and smooth; haemolysis is usually absent. Microscopically, the cells are short Gram-negative rods with rounded ends; vacuoles may be present when cultured in media containing a fermentable carbohydrate. The B. melaninogenicus - B. oralis group as well as B. asaccharolyticus comprise the black pigmentproducing species, the pigment becoming evident after prolonged incubation (3-5 days) on Blood Agar plates. In young cultures (1-2 days) the species in this group produce a brick-red fluorescent pigment (protoporphyrin) visible under long-wave ultraviolet light (Wood's lamp, 365 nm); fluorescence disappears as colonies develop their brown-black pigmentation (protohaemin). Microscopically, the cells are usually coccobacillary in morphology. B. ureolyticus (formerly B. corrodens) has a distinctive 'pitting' colonial morphology on solid media owing to 'corrosion' of the agar surface. Pitting colonies may only become apparent after incubation for several days, particularly when isolated in mixed

[7.2] culture, as is usual with this species. Colonially, B. ureolyticus may be confused with the capnophilic facultative anaerobe Eikenella corrodens (Section 7.10.3). The former is readily distinguished by urease production, metronidazole sensitivity and gelatinase production. Moreover, the growth of B. ureolyticus is enhanced by supplementing media with sodium formate and fumaric acid. B. splanchnicus, an uncommon clinical isolate, may be confused initially with members of the B. fragilis group since it is resistant to aminoglycosides and grows well on bile-containing media. This species is distinguished by non-fermentation of sucrose although gas chromatography provides better differentiation. B. disiens, B. bivius and B. oralis are easily confused with B. fragilis but growth in the presence of 20% bile effectively separates them. Moreover, B. disiens and B. brevis do not ferment sucrose. Mi redefinition: Bacteroides. Gram-negative rods (may be coccobacillary); nonsporing; usually non-motile. Anaerobic; sensitive to metronidazole. Fermentative or non-fermentative. Type species: B. fragilis; NCTC strain 9343. 7.2.2 Fusobacterium (Table 1.2b). Many of the species currently included in this genus are insecure, and only Fusobacterium nucleatum (the type species) is consistently fusiform (spindle-shaped) in cellular morphology. Other species may be pleomorphic, or difficult to distinguish microscopically from Bacteroides. Colonies of many strains on Blood Agar fluoresce yellow-green in long-wave ultraviolet light (365 nm). The species commonly encountered in clinical material are sensitive to kanamycin (1000 |ig disc) and colistin (10 jig disc), but resistant to vancomycin (5 jig disc). Strains of F. necrophorum are usually haemolytic on horse Blood Agar, and many strains are lipase-positive, showing restricted opalescence and a pearly layer on Egg-yolk Agar (Holdeman & Moore, 1972). We show other differential reactions for the few strains encountered most frequently in clinical material in Table 1.2b. We do not, however, include the morphologically large organism of Vincent's angina, previously classified as 'Fusiformis fusiforme' and now transferred to the genus Leptotrichia as the only species, L. buccalis, 97

CHARACTERS OF GRAM-NEGATIVE BACTERIA

Table 7.2b. Second-stage table for Fusobacterium

Indole Aesculin Growth in 20% bile Carbohydrates, acid from: glucose fructose lactose mannose Lipase Propionate from lactate* Propionate from threonine* 1 2 3 4

Fusobacterium Fusobacterium Fusobacterium Fusobacterium

+ -

+ -

+ +

d +

— d + +

-

+ + + -

+ + .,— + -

+

+

— -

+

+

necrophorum nucleatum mortiferum varium

*For laboratories with gas chromatography facilities Symbols used in the Table are explained in Tables 5.1 and 5 .'2 on p.47.

which has similar growth requirements. The identification of fusobacteria in routine laboratories is described by Bennet & Duerden (1985). Minidefinition: Fusobacterium. Gram-negative rods (may or may not show typical fusiform morphology); non-sporing; nonmotile. Anaerobic; sensitive to metronidazole. Non-fermentative or weakly fermentative. Type species: F. nucleatum; ATCC strain 2556. 7.2.3 Veillonella, Addaminococcus, Megasphaera (Table 7.3). As with Bacteroides and Fusobacterium, the identification and classification of Veillonella and the two related genera of Gramnegative anaerobic cocci, Addaminococcus and Megasphaera, were beset with difficulties (Rogosa, 1971). Technically, there was considerable doubt previously about their purity as many cultures were mixed and probably growing synergistically; and they were extremely intolerant of oxygen, dying out rapidly on minimal exposure to air at the bench. Poor and/or conflicting descriptions occurred frequently but, by using anaerobic cabinets and other procedures to maintain strict anaerobiosis and by means of gasliquid chromatography, a number of different genera and species were defined and are currently accepted as valid (Rogosa, 1984). The three genera recognized 98

[7.2]

are all members of the normal flora of the oropharynx and gastrointestinal tract of man. They are rarely encountered in clinical material and their pathogenic significance, if any, is low; we do not therefore give a second-stage table for species identification but we show the generic characters in Table 7.3 for comparison with those of other Gram-negative cocci. Like fusobacteria, they are resistant to vancomycin (5 jug). Veillonella parvula is the commonest isolate from human material; other species include V. atypica and V. dispar. Definitive identification of the genus and species requires gas-liquid chromatography of metabolic end products, and a range of biochemical tests. However, Megasphaera is distinguished from the other two genera by its large cell size (>2 \\m) and ability to ferment glucose, fructose and maltose. Minidefinition: Veillonella. Gram-negative cocci: non-motile; non-sporing. Anaerobic; sensitive to metronidazole. Non-fermentative. Type species: V. parvula; NCTC strain 11810. 7.3

The Gram-negative aerobic cocci (Branhamella, Neisseria,) Characters common to members of the group'. Gram-negative cocci or coccobacilli. Non-motile. Aerobic.

Apart from one species, Neisseria elongata, which forms long filaments under the influence of penicillin, all other members of the genus Neisseria and those of Branhamella (which were originally placed in Neisseria) as well as those of the anaerobic genus Veillonella are all Gram-negative cocci - a morphological characteristic which they share with the coccal forms of Acinetobacter. To American bacteriologists, acinetobacters were originally regarded as 'mimeas', a group of poorly-described Gram-negative cocci that could be confused with gonococci. The morphological resemblance is real, especially in direct smears from clinical specimens, but in culture the acinetobacters soon become slightly elongated, coccobacillary and finally assume a true rod-like form. As clinical bacteriologists can encounter the coccal form of acinetobacters we include them in Table 7.3 for comparison with branhamellas and neisserias. The characters of acinetobacters are

[7.3]

GRAM-NEGATIVE AEROBIC COCCI

Table 7.3. Second-stage table for Acinetobacter, Branhamella, Gemella, Neisseria and Veillonella 1 Gram reaction Aerobic Growth Catalase Oxidase Carbohydrate breakdown [F/O/-] Pigment production Haemolysis Growth at 22 °C Growth on Nutrient Agar Requirement for blood or serum Carbohydrates, acid from: glucose lactose maltose sucrose Nitrates reduced

+ + + + O Ob -/p -/p + + + + +a da -

+ + + w +

F

-

6

7

8

9

10

de

+

+

+

+

+

+

+

+

+

_

O* O* Qb

-

-

d

-

de

1 Acinetobacter calcoaceticus; 'Acinetobacter anitratus'; 7 'Moraxella Iwoffii var. glucidolytica'; M. glucidolytica'; 8 'Herellea vaginicola' 2 Acinetobacter Iwoffii; 'Moraxella Iwoffii'; 9 'Mima polymopha' 10 3 Branhamella catarrhalis; 'Neisseria catarrhalis' 11 4 Gemella haemolysans; 'Neisseria haemolysans' 12 5 Neisseria cinerea 6 Neisseria elongata 13

12

+ +

ob o^

+8

dc -c

11

13

D +

O*

9 _

-

-J

Neisseria flavescens Neisseria gonorrhoeae; gonococcus Neisseria lactamica Neisseria meningitidis; W. intracellularis'; meningococcus Neisseria mucosa; 'Diplococcus mucosus' Neisseria subflava; N. flava; N. perflava; N. sicca Veillonella spp.

a b c d e

Positive also in glucose Peptone Water and lactose (10%) in Nutrient Agar. No change, or alkali produced, in the open tube of Hugh & Leifson's medium. Negative in glucose Peptone Water and lactose (10%) in Nutrient Agar. Gram-positive but easily decolorised. Strains showing no catalase activity and no production of acid from glucose correspond to N. elongata subsp. elongata; strains producing catalase and weak acidity from glucose correspond to N. elongata subsp. glycolytica. f Negative on isolation; subcultures may give positive reactions. g On first isolation; many strains will grow on Nutrient Agar on sub-culture. h May be positive in some media. Other symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

shown again in Table 1 Aa with the kingellas and moraxellas and also some other phenotypically similar Gram-negative rods. At the generic level, veillonellas do not cause problems in identification as their strictly anaerobic habit readily distinguishes them from acinetobacters, branhamellas and neisserias, all of which fail to grow anaerobically; we deal with Veillonella in Section 7.2.3 but include it also in Table 7.3 to show some characters of all the Gram-negative cocci together. Although not really a member of this group, Gemella

haemolysans was first described as a neisseria and as it usually appears to be Gram-negative, we include it in Table 7.3 for comparison with Neisseria species. It is, however, a weakly Gram-positive organism and its rightful place is in Chapter 6 (Section 6.3.4). 7.3.1 Neisseria (Table 7.3). In this genus, many species have been described and, when the lack of reactivity in biochemical tests is taken into account, this at first seems unwarranted. Molecular biological and genetic techniques, however, have confirmed the 99

CHARACTERS OF GRAM-NEGATIVE BACTERIA

heterogeneous nature of the constituent species of Neisseria and indicated that the genus as circumscribed in the late 1960s needed revision (Kingsbury, 1967; Henriksen & B0vre, 1968Z?; Baumann, Doudoroff & Stainer, 1968a; Kingsbury et al, 1969; B0vre, Fiandt & Szybalski, 1969). Subsequently Catlin (1970) proposed that 'Neisseria catarrhalis' should form the type species of a new genus, Branhamella, and she thought that other species might follow because N. caviae and N. ovis, by gasliquid chromatography, form a homogeneous group with Branhamella catarrhalis (Lambert et al., 1971). Genetic studies have confirmed the close relationship of these two species to each other and to a third species, N. cuniculi (B0vre, 1980; B0vre & Hagen, 1981). Though not all taxonomists would include these three species of so-called 'false neisseriae' in the same taxon as the catarrhalis organism, it is nevertheless convenient to consider them together as a single taxon. B0vre (1979) proposed that this taxon should be regarded not as a genus but as the subgenus Branhamella of the genus Moraxella in order to reflect the closer relationship of these organisms to each other than to the 'true neisserias'. The close relationship of Branhamella to Moraxella (and to Acinetobacter) and their lack of relatedness to the 'true neisserias' have now been confirmed by rRNA-DNA studies (Rossau et al, 1986). Of the species placed in the sub-genus Branhamella, only the catarrhalis organisms is likely to occur in clinical specimens and it is therefore the only organism in this group that we show in Table 7.3. We continue to refer to it by the approved name Branhamella catarrhalis rather than Moraxella (Branhamella) catarrhalis as proposed by B0vre (1979). Neisseria flavescens, a species thought to have caused a limited epidemic of meningitis in Chicago (Branham, 1930), has rarely been isolated since. Prentice (1957) thought that the organism he isolated from a patient with meningitis might be N. flavescens and Wertlake & Williams (1968) described the organism from a case of septicaemia. At first none of the Chicago isolates attacked any sugars but after many years of laboratory propagation, the strains originally received from Branham and deposited in the National Collection of Type Cultures, London, as well as those she kept herself, all developed the ability to produce acid from glucose, maltose, and 100

[7.3]

sucrose. We accordingly include N. flavescens in Table 7.3 but show its original characters in the (faint) hope that it may be isolated again and recognized. The exclusion of W. catarrhalis', N. caviae, N. cuniculi and N. ovis from Neisseria leaves a genus (excluding N. cinerea and N. elongata subsp. elongata) composed of bacteria that can attack carbohydrates in suitable media; as usually described, N. flavescens is said not to attack carbohydrates but the truth of this statement seems doubtful, though the ability may be masked on first isolation. In the minidefinition we therefore exclude species that do not attack any sugars. Neisserias grow better on the surface of solid media than in equivalent liquid media; most strains grow better in an atmosphere with increased CO2 and it is doubtful if any will grow under strictly anaerobic conditions. They are not easily characterized as freshly isolated strains grow feebly in artificial culture. Some species are described as Gram-variable or are said to resist decolorization; this may seem to be a difference due to technical variation in different laboratories, but it is reported so frequently that notice should be taken of it. Henriksen & B0vre (1968b) described the tendency to resist decolorization as a characteristic of the family Neisseriaceae which, in their view at that time, comprised only the genera Moraxella and Neisseria though it now also includes Acinetobacter, Branhamella and Kingella (but see Rossau et al., 1986). Bacillary forms may occur in N. elongata (B0vre & Holten, 1970) and, judged by the G+C content of DNA and other genetic information, the generic allocation is correct. The pathogenic neisserias grow feebly or not at all in media prepared for the usual biochemical characterization tests, and are thus worthy candidates for the rapid single-substrate tests in which multiplication of the organism is not essential, the reaction occurring between preformed enzymes and the substrate without interference from metabolic by-products. The substrate Af-y-glutamyl-pnaphthylamide has been used for distinguishing gonococci from meningococci (D' Amato et al., 1978; Hoke & Vedros, 1982) and commercial rapid test kits based on this principle are now available for identification of neisserias. It is often impossible to decide, on purely laboratory information, whether an isolate is a gonococcus or a meningococcus; the sugar reactions of these

GRAM-NEGATIVE AEROBIC COCCI

organisms may be unreliable due to poor growth and, although most meningococci will produce acid from both glucose and maltose, some undoubted meningococci may attack glucose only and, occasionally, neither sugar. More reliable results may be obtained with the rapid sugar fermentation test described by Vedros (1978) which also obviates the need for growth by relying on preformed enzymes. Most neisserias do not produce acid from lactose, but Mitchell, Roden & King, (1965) collected thirty-five lactosepositive strains of meningococcus-like bacteria over a period of fifteen years. Hollis, Wiggins & Weaver, (1969) named these organisms 'Neisseria lactamicus ', since corrected to N. lactamica. The species Neisseria animalis (Berger, 1960b), TV. canis and TV. denitrificans (Berger, 1962) as well as those now included in Branhamella - N. caviae (Pelczar, 1953), N. cuniculi (Berger, 1962) and N ovis (Lindqvist, 1960) - are excluded from Table 7.3 as they have not been reported from clinical specimens. In Table 7.3 we show the classical nasopharyngeal commensals, N. flava, N. perflava and N. sicca under the umbrella species Neisseria subflava (previously collected together as W. pharyngis') as there seems little to be gained from maintaining their independent status. Minidefinition: Neisseria. Gram-negative cocci. Aerobic; catalase-positive; oxidasepositive. Attack sugars by oxidation. Type species: N. gonnorrhoeae; NCTC strain 8375. 7.3.2 Branhamella (Table 7.3). This genus was created by removing from Neisseria those species which differed significantly in the base composition of their DNA: in Branhamella the G+C mol % value varies between 40 and 44, and in Neisseria it is about 50. The two genera also differ in the fatty acid composition of the bacterial cells (Catlin, 1970). The lack of a close relationship between these two genera is further confirmed by rRNA-DNA hybridization studies (Rossau et aL, 1986). Unfortunately these characteristics are not among those on which generic differentiation depends in diagnostic laboratories but, as shown in Table 7.3, the distinction can be made with tests that are part of the normal routine. We show only B. catarrhalis in Table 7.3 as the other three species have not been reported from man. Like neisserias, but possibly even more so, the

[7.3] bacterial cells of the branhamellas tend to resist decolorization when stained by Gram's method. In contrast, Gemella, a Gram-positive coccus with the opposite characteristic (a Gram-positive organism that is easily over-decolorized), is shown in Table 7.3 so that its characters can be compared with those of Branhamella catarrhalis and Neisseria species. Of the usual characterizing tests, those for the reduction of nitrate and the production of acid from carbohydrates are among the most useful. With Branhamella catarrhalis, the former test is often positive and the latter negative; the other species left in Neisseria (excluding N. cinerea, N. elongata subsp. elongata, N. flavescens and N. mucosa) fail to reduce nitrate and all attack carbohydrate(s) (but see Section 7.3.1). Minidefintion: Branhamella. Gram-negative cocci which may resist decolorization. Aerobic. Catalase-positive; oxidase-positive. Do not produce acid from carbohydrates. Generally reduce nitrate to nitrite. Type species; B. catarrhalis; NCTC strain 11020. 7.3.3 Acinetobacter (Tables 7.3; 7.4a) is a genus in which two species are now recognized (but see Section 7.4.2) in contrast to the 17 listed by Prevot (1961). These two species have a coccal or coccobacillary morphology, and in smears may show a superficial resemblance to the gonococcus or meningococcus. In a series of papers, de Bord (1939, 1942, 1943, 1948) rightly emphasized this morphological similarity but confused the picture by describing briefly (and inadequately) several different organisms, including two new species of Neisseria; and, except for the two new neisserias, he placed them all in a new tribe which he named Mimeae. One of the species that mimicked the gonococcus morphologically is now known as Acinetobacter calcoaceticus (formerly 'A. anitratus') and this species (together with Acinetobacter Iwoffii) is shown in Table 7.3 for comparison with Neisseria gonorrhoeae and N. meningitidis. The acinetobacters have much in common with the moraxellas and recent rRNA-DNA hybridization results (Rossau et aL, 1986) lend further support to previous suggestions that the two genera should be united; both genera are shown in greater detail in Table lAa. 101

CHARACTERS OF GRAM-NEGATIVE BACTERIA

Minidefinition: Acinetobacter. Non-motile, Gram-negative coccobacilli or short rods. Aerobic. Catalase-positive; oxidase-negative. Attack sugars by oxidation or not at all. Do not produce pigment. Arginine test negative. Type species: A. calcoaceticus; ATCC strain 23055. 7.3.4 Gemella (Tables 6.3 a, c; 7.3), an organism that is easily decolorized and usually appears to be Gram-negative (as it was first described). In cell structure and mode of division it resembles the Gram-positive bacteria (Reyn, 1970) and in its cultural characters it seems to be close to the streptococci. We describe this organism with the Gram-positive cocci in Table 6.3a, c and Section 6.3.4, but because of the ease with which it is decolorized, we also show it with the Gram-negative cocci in Table 7.3. Minidefintion: Gemella. Gram-positive cocci (but easily decolorized). Aerobic; facultatively anaerobic. Catalase-negative; oxidase-negative. Attack sugars by fermentation; do not produce gas. Type species: G. haemolysans; ATCC strain 10379. 1A

The Moraxella-Acinetobacter group: the non- (or weakly) saccharolytic non-motile rods {Moraxella, Acinetobacter, Kingella, Brucella, Bordetella) Characters common to members of the group: Gram-negative coccobacilli or short rods, non-motile (with the exception of Bordetella bronchiseptica). Strictly aerobic. Catalase-positive. Do not oxidize many carbohydrates.

There have been suggestions that the genera Moraxella and Acinetobacter should be combined, and it is true that they have much in common, hence the inclusion of both in the family Neisseriaceae (together with Branhamella, Kingella and Neisseria). Indeed, species have been transferred from one genus to the other (for example 'Moraxella iwoffiV to Acinetobacter Iwoffii) and confusion has arisen because a species and its 'variety' were equated with species in different genera {'Mima polymorpha" = Acinetobacter Iwoffii and 'Mima polymorpha var. 102

[7.3]

oxydans" - Moraxella nonliquefaciens or Moraxella osloensis). Both Moraxella and Acinetobacter were created for species discarded from other genera. Organisms wrongly placed in the genus Haemophilus (because they were not haemophilic) were transferred to Moraxella; and the non-motile members of Achromobacter, itself formerly an unsatisfactory collection of non-pigmented Gram-negative rods, were transferred to Acinetobacter. Achromobacter is dealt with in Section 7.6.1. 7.4.1 Moraxella (Table 7.4a) was created by Lwoff (1939) for non-haemophilic bacteria that had previously been placed in Haemophilus; these bacteria did not need X and/or V factor(s), and did not attack carbohydrates. Most of the organisms placed by Lwoff in this genus had been isolated from the conjunctiva, but other workers added strains from other sources ('M. Iwoffii' from soil) and also strains that attacked carbohydrates CM. IwoffiV var. glucidolytica'); the latter organism was subsequently shown to be 'Bacterium anitratum" (Brisou & Morichau-Beauchant, 1952), later known as 'Acinetobacter anitratus" and currently as Acinetobacter calcoaceticus. Brisou (1953), Floch (1953) and others excluded 'M. IwoffiV and the sugar-attacking species from Moraxella; Henriksen (1960) also excluded 'M. IwoffiV and restricted the genus to oxidase-positive organisms. The debate about what should be included and what excluded from the genus was prolonged (it may not have ended yet) and complicated, but most workers (see Baumann, Doudoroff & Stanier, 1968a; Henriksen & B0vre, 1968£; Samuels et al., 1972; Lautrop in Bergey's Manual (1974) agree that only oxidase-positive, non-saccharolytic organisms should go into Moraxella. An organism resembling Moraxella lacunata was isolated from the conjunctiva of guinea-pigs (Ryan, 1964), and Moraxella bovis causes conjunctivitis in cattle and horses (strains from horses, formerly regarded as a separate species, M. equi, are now included in M. bovis (B0vre, 1984)). Although Moraxella bovis has not been reported from human clinical material, it is retained in Table 1 Aa for completeness. Scandinavian workers report that it is unable to reduce nitrates and that the catalase test is positive; however, in the USA, Pugh, Hughes &

MORAXELLA-ACINETOBACTER

GROUP

[7.4]

Table 7.4a. Second-stage table for Acinetobacter, Bordetella, Brucella, Kingella and Moraxella 1

2

3

4

5

6a

7

8

Catalase

+

+

+

+

+

"

+

+

Carbohydrate breakdown [F/O/-] PHBA* accumulation in cells Haemolysis on Blood Agar Growth at 42 °C Growth on Nutrient Broth/Agar Growth on MacConkey Citrate as C source Carbohydrates, acid from: glucose ethanol maltose xylose Nitrate reduced to nitrite Nitrite reduced Gelatin liquefaction Urease Tween 20 hydrolysis Tween 80 hydrolysis Brown pigment on Tyrosine Agar Growth on 40% bile Growth stimulation by bile Growth on 4% NaCl

O -

O^ _ -/P d + + + d d _

_

_ -

_ d d _ + + + d d d

-/p d + +

+c + + d d + + + -

dd d d d + + + d d

+ + ? ? -

9 9 9 ?

7 7 7 7 7 7 _ 7 7

-

- -/p P

d + d _

+ _

_ -

_e -

-

(+)

+ d

-/ -

d

d

d

-

+ -

-

-+

_ d + + d _ -

9

10

11

+

+

_ d -

_ + + d _

_

-

d -

-8

-8

-

+ d _ d

d d^ d d d

+ -

+ + _ -

d + d + +

12

_ + + + + d + + +

1 Acinetobacter calcoaceticus; lA. anitratus'; 'B5W; 'Moraxella Iwoffii var. glucidolytica'; 'M. glucidolytica'; 'Herellea vaginicola' 2 Acinetobacter Iwoffii; 'Moraxella Iwoffii'; 'Mima polymorpha' 3 Bordetella pa rape rtussis; 'Haemophilus parapertussis''; 'Alcaligenes parapertussis' 4 Bordetella pertussis; 'Haemophilus pertussis' 5 Brucella spp; B. melitensis'('Alcaligenes melitensis'; 'Brucella bruceV); B. abortus '(Bang's bacillus); B. suis 6 Kingella kingae; 'Moraxella kingiV 7 Moraxella bo vis; 'M. equi' 8 Moraxella lacunata; 'M. duplex'; 'M. liquefaciens' 9 Moraxella nonliquefaciens; 'Mima polymorpha var. oxidans' 10 Moraxella osloensis 11 Moraxella phenylpyruvica 12 Moraxella urethralis * a b c d e / g h P

PHBA: Poly-P-hydroxybutyrate Biochemically similar strains producing indole correspond to K. indologenes. No change, or alkali produced, in the open tube of Hugh & Leifson's medium. Positive in glucose Peptone Water. Negative in glucose Peptone Water. Positive on Ascitic Agar + glucose. Biochemically similar strains reducing nitrate correspond to K. denitrificans. May be positive when carried out as a buffered single substrate test. Some strains positive on first isolation; the property is soon lost. Clear zone of haemolysis around colonies.

Other symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

103

CHARACTERS OF GRAM-NEGATIVE BACTERIA

McDonald, (1966) found that all their strains reduced nitrates and were catalase-negative. This perhaps represents an example of 'geographical races' of species. Moraxella phenylpyruvica (B0vre & Henriksen, 1967) deaminates phenylalanine and tryptophan when suitable methods are used, as do several other Moraxella species; the test of Shaw & Clarke (Appendix C3.45) is not sufficiently sensitive and Snell & Davey (1971) recommend the method of Goldin& Glenn (1962). Of two organisms provisionally placed in the genus Moraxella, one, 'M. urethralis (Lautrop, B0vre & Frederiksen, 1970), was said to resemble M. osloensis. However, more recent DNA data (B0vre, 1984; Rossau et ah, 1986) place M. urethralis well apart from the other major taxa and suggest that it is a candidate for a new genus within the family Neisseriaceae. It has since been proposed that a new genus, Oligella, should be created for this organism as well as for an Alcaligenes-like organism formerly known as Group IVe (Rossau et al., 1987). The other organism, 'M. kingiV (Henriksen & B0vre, 1968a), is saccharolytic on media enriched with ascitic fluid, and is now placed in the genus Kingella (Section 7.4.3). The proposal by Henriksen & B0vre (1968b) to include the so-called 'false neisserias ' ('Neisseria catarrhalis\ N. caviae, N. cuniculi and N. ovis) in the genus Moraxella has not been followed in this Manual, 'N. catarrhalis" will be found in Branhamella (Section 7.3.2). Cultures of Moraxella species are sensitive to drying and some strains will not grow at 37 C unless they are in a moist atmosphere; they grow best when incubated in a closed jar (Henriksen, 1952). Minidefinition: Moraxella. Gram-negative rods; non-motile. Aerobic. Catalase-positive; oxidase-positive. Sugars not attacked. Growth improved by the addition of blood or serum but specific growth factors are not known. Type species: M. lacunata; NCTC strain 11011. 7.4.2 Acinetobacter (Tables 7.3; 7.4a). A genus proposed by Brisou & Prevot (1954) for non-motile species that, apart from their lack of motility, would have fitted into Achromobacter as defined at that time; they included in it, as 'Acinetobacter 104

[7.4]

anitratum" (sic), two species, 'Bacterium anitratum" (Schaub & Hauber, 1948) and 'Moraxella Iwoffii var. glucidolytica," which had previously been shown to be identical (Brisou & Morichau-Beauchant, 1952). In 1961, Prevot listed 17 species in the genus, but as a taxonomic entity Acinetobacter has had a chequered career, with much debate about the species to be included, and whether it should be a separate genus or included in Moraxella. The first good descriptions of organisms in the taxon were published by Schaub & Hauber (1948) for 'Bacterium anitratum" and by Stuart et al. (1949) for the 'B5W group'. Ewing (1949a) recognized that these organisms bore some resemblance to the poorly described tribe Mimeae with genera 'Mima\ 'Herellea\ and 'Colloides' (De Bord, 1939, 1942, 1943, 1948), some strains of which simulated gonococci. Henriksen (1963) and Pickett & Manclark (1965) showed that these names were being misapplied and that because the original descriptions had been vague and ambiguous, considerable confusion had resulted; these names are now no longer in use. Steel & Cowan (1964) modified the definition of the genus Acinetobacter to allow inclusion of bacteria that did not produce acid from carbohydrates; this was an improvement and allowed the inclusion of 'Moraxella iwojfiV, an organism that was misplaced in Moraxella. But they were imprudent in suggesting also the transfer to Acinetobacter of the glanders bacillus, which has since found a better resting place in the genus Pseudomonas. After considerable bibliographical research, Baumann, Doudoroff & Stanier, (1968Z?) found a reference to an organism labelled 'Micrococcus calcoaceticus' which had the characteristics of 'Acinetobacter anitratus\ and thus added yet another name. Because it is the oldest, calcoaceticus becomes the nomenclaturally correct specific epithet thus producing the awkward but currently approved combination Acinetobacter calcoaceticus. Juni (1984) included both A. calcoaceticus and A. Iwoffii in the same species under the former name; however, we prefer to distinguish the two species as it is not difficult to do so and it may also be helpful clinically. Indeed it was known for some time that Acinetobacter was heterogeneous both nutritionally (Baumann, Doudoroff & Stanier, 1968/?, see below) and genetically (Johnson, Anderson & Ordal, 1970). The genus is now known

[7.4]

MORAXELLA-ACINETOBACTER GROUP

to comprise more than 12 genetically distinct species, including A. calcoaceticus and A. Iwojfii; four of the remaining species have been named A. baumannii, A. haemolyticus, A. johnsonii and A. junii (Bouvet & Grimont, 1986). However, as the phenotypic distinction between these species depends almost entirely on nutritional characters, we have not tried to include them in the Tables. One of the characteristics of Acinetobacter calcoaceticus is its ability to attack monosaccharides but not higher saccharides in Peptone Water sugars. When the sugar concentration is raised to 5 or 10%, the organism can attack lactose. A non-specific aldose dehydrogenase (Baumann, Doudoroff & Stanier, 1968b) is responsible for the oxidative production of acid from several carbohydrates in complex media. As only one enzyme is involved, the pattern of carbohydrates oxidised is of no value for classification though it is still useful for identification. Biotypes of Acinetobacter calcoaceticus occur and these may vary in soap tolerance (Billing, 1955), gelatin liquefaction, and ability to grow at 44 °C (Ashley & Kwantes, 1961). Several biotypes were described by Baumann, Doudoroff & Stanier, (1968Z?); some of these may perhaps correspond to distinct genospecies The nutritional tests necessary for recognition of 12 of the genospecies can be used also for biotyping purposes (Bouvet & Grimont, 1987). In the characterization of Acinetobacter species there is general agreement on the main points but some disagreement on detail; urease, for example, has been reported as negative, variable, and positive for the two species we recognize here. The differences may be explained by Henderson's (1967) finding that urease production by members of this genus is suppressed by ammonia produced from peptone and other nitrogenous constituents of the media. Minidefinition: Acinetobacter. Non-motile, Gram-negative coccobacilli or short rods. Strictly aerobic. Catalase-positive; oxidasenegative. Attack sugars by oxidation or not at all. Do not produce pigment. Arginine test negative. Type species: A. calcoaceticus; ATCC strain 23055. 7.4.3 Kingella (Table 7.4a) is a genus originally proposed to accommodate the catalase-negative 'Moraxella' species, 'M. kingiV. On transfer

Table 7.4b. Third-stage table to distinguish the classical 'species'
C0 2 requirement H2S produced Growth in: thionin^ 1/25 000 thionin* 1/50 000 basic fuchsin 1/50 000 safranin O 1/5000

+ + (early)

-

Da

+ d

+ + +

+ +

1 Brucella abortus; Bang's bacillus 2 Brucella melitensis; B. brucei; Alcaligenes melitensis 3 Brucella suis a American strains positive; produce H2S over several days. Danish strains do not produce H2S. See note in text on geographical races. b Dyes obtained from the National Biological Stains Department, Allied Chemical Corporation, New York, USA. Other symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

(Henriksen & B0vre, 1976) the name was corrected to Kingella kingae. Soon afterwards two other species were added to the genus (Snell & Lapage, 1976); one reduces nitrate and generally nitrite (K. denitrificans); the other produces indole (K. indologenes). Genetic studies demonstrate a close relationship between the 'true neisserias', K. kingae and K. denitrificans, whereas K. indologenes is not closely related to them (Rossau et ai, 1986). Minidefinition: Kingella. Non-motile, Gram-negative short rods. Aerobic or facultatively anaerobic. Catalase-negative; oxidase-positive. Attack sugars by fermentation in suitable media. Do not produce pigment. Arginine test negative. Type species: K. kingae; NCTC strain 10529. 7.4.4 Brucella (Tables 7.4a, b) comprises a group of bacteria whose cultural and serological characters blend into each other so that some workers regarded the whole as one species, while others saw them as three not-too-well-defined species; others accepted the three species without question and were willing to consider other candidates for inclusion in the genus. In 1963 the international Brucella Subcommittee (Stableforth & Jones) decided in favour of three species, each divided into biotypes, and then accepted 105

CHARACTERS OF GRAM-NEGATIVE BACTERIA

a new species, Brucella neotomae, only seven strains of which had then been isolated. Other species were later proposed and B. ovis and B. canis were provisionally accepted by the Subcommittee (Jones & Wundt, 1971). More recent DNA studies (Verger et al, 1985) show that the genus does, in fact, comprise a single genospecies, B. melitensis. We continue, however, to use the 'old' specific epithets although strictly they should be used in a vernacular form for biovar designation (e.g. Brucella melitensis biovar Abortus 1). The generic characters are shown in Table lAa, and some distinguishing features of the three classical species Brucella abortus, B. melitensis, and B. suis are shown in Table 1.4b; details of the various biotypes are given in the report of the Brucella Subcommittee (Jones & Wundt, 1971), and the technical methods are described by Morgan & Gower (1966). The tests used to separate the biotypes include investigation of the oxidative metabolism of isolates (Meyer & Cameron, 1961a, b), the sensitivity to Tbilisi bacteriophage (which is not specific for any 'species') (Parnas, 1961), and serological tests with monospecific antisera (which are not available commercially and are not easy to prepare). Differentiation kits and the fluorescent antibody reagents available commercially are not adequate for distinguishing between the various biotypes, and their use does not give better results than the tests shown in Table 1 Ab. The performance of the latter tests must be well controlled at all stages and, since their interpretation is subjective, they can be relied upon only when strains are examined routinely as in a reference laboratory. In Table lAb we show H2S production by Brucella suis as 'D', indicating differences between the main biotypes; American strains produce H2S and Danish strains do not. These may be more examples of 'geographical races' of species. Brucella neotomae differs from the classical brucellas in that acid production from various sugars can easily be shown in peptone-containing media and it is oxidase negative. Brucella ovis is also oxidase negative; it needs CO2 for isolation and does not produce H2S or reduce nitrates. We do not show these two 'species' in the tables. Except for Brucella neotomae, brucellas do not produce acid from carbohydrate in media containing 106

[7.4]

peptone, but by avoiding the peptone, acid production from carbohydrate can be shown in the classical Brucella 'species' (Pickett & Nelson, 1955). Minidefinition: Brucella. Short Gram-negative rods; non-motile. Aerobic or carboxyphilic. Catalase-positive; usually oxidase-positive. Do not show acid production from sugars in peptone-containing media (except for one 'species'). Urease-positive. Type species: B. melitensis; NCTC strain 10094. 7.4.5 Bordetella (Tables 7.4a; 7.5). Apart from clinical association, the species within this genus appear to have few characters in common. The whooping cough organism, Bordetella pertussis, does not grow on ordinary media until it has been in laboratory culture for some time, and a medium rich in blood has usually been considered essential for its isolation. Bordet-Gengou is probably the medium most often chosen, and many attempts have been made to improve its selectivity (see, for example, Lacey, 1951). Its nutritional requirements and metabolism were reviewed by Rowatt (1957), and its isolation and identification by Lautrop (1960). Though the whooping cough organism is nutritionally exacting, Turner (1961) showed that it does not require a rich medium; peptones, however, contain substances that are inhibitory to B. pertussis, though a meat extract agar without peptone is suitable for isolation of the organism. The other two species in the genus, B. parapertussis and B. bronchiseptica, though also associated with infections of the respiratory tract, are much less exacting in their nutritional requirements. B. pertussis is non-motile and oxidasepositive, B. parapertussis non-motile and oxidasenegative, and B. bronchiseptica is motile and oxidase-positive. This diversity of primary characters makes it difficult to define the genus and it is small wonder than B. parapertussis and B. bronchiseptica have each resided in various other genera in the past. In conventional biochemical tests B. bronchiseptica is more similar to Alcaligenes faecalis than to the other two Bordetella species. However, despite the heterogeneity of these characters in the genus, other considerations such as DNA homology confirm that phylogenetically the three species should be included in the same genus.

MORAXELLA-ACINETOBACTER GROUP

Mi redefinition: Bordetella. Gram-negative rods; may be motile by peritrichous flagella or non-motile. Aerobic. Catalase-positive; oxidase may be positive or negative. One species (B. pertussis) does not grow in simple media containing peptone. Do not attack sugars. Type species: B. pertussis; NCTC strain 10739. 7.5

The non-(or weakly) saccharolytic motile rods {Alcaligenes, Shewanellas, alkali-producing pseudomonads) Characters common to members of the group: Gram-negative rods; motile. Strict aerobes. Catalase-positive; oxidase-positive. Produce acid from only a few carbohydrates.

The group is heterogeneous but all species come within the descriptive term motile and non-(or weakly) saccharolytic; they do not usually show acid production from glucose in ammonium salt medium. Most of the 'non-fermenters' by definition also fail to produce acid from carbohydrate in media containing peptone, though without it some of them are actively saccharolytic. It is essential to check the nitrate medium for residual nitrate after a negative nitrite test as the test for nitrite can be negative when nitrate has been reduced, and the test for residual nitrate is an essential part of the nitrate reduction test (see Table 4.1). A feature of the group is its lack of positive characters; rarely, there is an absence of information, shown by question marks in the tables. This absence is understandable for organisms such as Bordetella pertussis which are unable to grow in or on the media used for so many tests, but is is surprising for those organisms in this group which grow readily. Johnson & Sneath (1973) described a wide range of tests useful for characterizing these bacteria. 7.5.1 Alcaligenes (Table 7.5). Although ten species of Alcaligenes appeared in the Approved Lists of Bacterial Names (1980), previous studies have shown that the five marine species and three hydrogen-utilizing species are not closely related to the type species Alcaligenes faecalis. All the marine

[7.4] species except A. aquamarinus have now been transferred to a new genus, Deleya (Baumann, Bowditch & Baumann, 1983). 'Alcaligenes odorans' (Malek, Radochova & Lysenko, 1963) was first isolated from stools and urine and named 'Pseudomonas odorans"\ descriptions of the odour have been typically subjective from valerian to jasmine - and from strawberry-like in young to ammoniacal in old cultures. A variant, 'Alcaligenes odorans var. viridans\ has a fruity smell and produces a bright green zone around colonies on Blood Agar (Mitchell & Clarke, 1965; Gilardi & Hirschl, 1969). However, the name 'A. odorans' no longer has any standing in nomenclature and it is now regarded as a synonym of A. faecalis (Kiredjian et aU 1981; Ruger & Tan, 1983; Holmes & Dawson, 1983). A. ruhlandii is an earlier, but less well-known synonym of Achromobacter xylosoxidans (Kiredjian et al, 1981; Holmes & Dawson, 1983). The latter organism and Alcaligenes denitrificans were not in the Approved Lists of Bacterial Names but both names have since been revived (Yabuuchi & Yano, 1981; Ruger & Tan, 1983) and these were the only two taxa retained with Alcaligenes faecalis in the genus Alcaligenes (Kersters & DeLey, 1984Z?). Although Achromobacter xylosoxidans is easily separated from Alcaligenes denitrificans (the former produces acid in ammonium salt medium from glucose and xylose whilst the latter fails to do so) other considerations show that they are closely related (Kersters & DeLey, 1984Z?). On the other hand, although A. faecalis and A. denitrificans are almost indistinguishable phenotypically (Table 7.5) they are not closely related genetically. It has been proposed that Achromobacter xylosoxidans should be transferred to the genus Alcaligenes as Alcaligenes xylosoxidans and that Alcaligenes denitrificans be made a subspecies, Alcaligenes xylosoxidans subsp. denitrificans (Kiredjian et al., 1986). Meanwhile, for practical identification purposes, we prefer to retain Achromobacter xylosoxidans in Achromobacter and Alcaligenes denitrificans in Alcaligenes together with Alcaligenes faecalis. A further species has been proposed: Alcaligenes piechaudii (Kiredjian et al., 1986) but it is not easily distinguishable from A. denitrificans by conventional biochemical tests and we do not include it in Table 7.5. Minidefinition: Alcaligenes. Gram-negative 107

[7.5]

CHARACTERS OF GRAM-NEGATIVE BACTERIA

rods; motile (by peritrichous flagella). Aerobic. Catalase-positive; oxidase-positive. Do not produce acid from sugars even in suitable media (some strains produce acid from ethanol). Type species: A. faecalis; NCTC strain 11953.

7.5.2 Shewanella (Table 7.5). The only species of this genus known to occur in clinical material (Holmes, Lapage & Malnick, 1975) is Shewanella putrefaciens. This organism was once included in Pseudomonas but was transferred because of its low DNA G+C content, which is characteristic of

Table 7.5. Second-stage table for Achromobacter, Alcaligenes, Bordetella, Shewanella and the alkaliproducing pseudomonads

Brown pigment Orange pigment Oxidase Nitrate reduced Simmons' citrate Christensen's citrate Urease Gelatinase production Growth in KCN medium H2S from TSI Ornithine decarboxylase Casein hydrolysis Deoxyribonuclease production Carbohydrates [in ammonium salt medium], acid from: glucose arabinose ethanol fructose glycerol maltose mannitol sucrose xylose Tween 20 hydrolysis Tween 80 hydrolysis Tyrosine hydrolysis Tyrosine, brown pigment production from Growth on b-hydroxybutyrate PHBA* accumulation in cells Growth on Cetrimide Agar Growth at 42 °C Lecithinase production 1 Achromobacter xylosoxidans; Alcaligenes xylosoxidans subsp. xylosoxidans 2 Alcaligenes denitrificans; Alcaligenes xylosoxidans subsp. denitrificans 3 Alcaligenes faecalis; 'A. odorans' 4 Shewanella putrefaciens; Alteromonas putrefaciens; 'Pseudomonas putrefaciens' 5 Bordetella bronchiseptica; 'Alcaligenes bronchisepticus'

1

2

3

4

5

6

7

8

9

10

11

_ + + + + d -

_ + +a d + + -

_ +

_ + + + + + + -

d _

d + d -

_ + + + d d + + d + +

_ + + + + d -

_ + + + + -

_ + + d -

_ + + d d d -

_ + d + + d d -

+ d d + + + + + d -

d _ + + d + d -

+ _ + + d + d -

d d _ d d + + d d + d +

-

+ -

-

+ + + d d d + d

d + d d -

_

+ + + + + + + + d -

d + d + + + -

+ + d d -

d + d + d d + -

6 Bordetella para pertussis; 'Haemophilus parapertussis'; 'Alcaligenes parapertussis' 7 Pseudomonas acidovorans 8 Pseudomonas alcaligenes 9 Pseudomonas diminuta 10 Pseudomonas pseudoalcaligenes 11 Pseudomonas testosteroni

* PHBA: Poly-p-hydroxybutyrate a Strains also reduce nitrite; strains failing to reduce nitrite correspond to Alcaligenes piechaudii. Other symbols used in the Table are explained in Tables 5.1 and 5.2 on p.47.

108

d + -

d + + + + + -

NON-(OR WEAKLY) SACCHAROLYTIC MOTILE RODS

Alteromonas (Lee, Gibson & Shewan, 1977). A more recent proposal is to transfer the organism once again, but to the new genus Shewanella (MacDonell & Colwell, 1985&). The species is somewhat heterogeneous and has been subdivided in various ways by different workers (Holmes, Lapage & Malnick, 1975; Owen, Legros & Lapage, 1978). We retain the organism as a single species which is easily recognisable by its salmon pink pigment, copious H2S production (positive even on Triple Sugar Iron agar - the only Gram-negative non-fermenter to do so) and decarboxylation of ornithine. Minidefinition: Shewanella. Gram-negative rods; motile. Aerobic. Catalase-positive; oxidase-positive. Some strains oxidize a few sugars. Type species: S. putrefaciens; ATCC strain 8071. 7.5.3 Pseudomonas: the alkali producers (Table 7.5). Although most pseudomonads produce acid from glucose in the open tube (only) of Hugh & Leifson's oxidation fermentation (O-F) medium (oxidative reaction) and from several carbohydrates in ammonium salt medium (Appendix A2.5.5), a few produce alkali in the open tube of glucose O-F medium and produce acid from only a few (or no) carbohydrates in ammonium salt medium. Such organisms, motile by polar flagella, may easily be confused with Bordetella bronchiseptica or Alcaligenes species unless flagellar staining is performed. The members of the group comprise P. acidovorans, P. alcaligenes, P. diminuta, P. pseudoalcaligenes and P. testosteroni. Their occurrence in clinical material is largely unknown as such strains are not easy to identify because of their non-reactive nature in conventional tests and they have probably often been misidentified in the past as Alcaligenes species. In the range of tests given in Table 7.5 the unnamed peritrichously flagellated organism 'Group IVc-2' (Clark et al., 1984) would be easily misidentified as P. testosteroni without flagella staining. However, the strains of Group IVc-2 we have examined differed from P. testosteroni in that none reduced nitrate, all produced urease, none produced acid from glycerol, all grew at 42 °C and many (78%) produced lecithinase. The revival of Comamonas terrigena proposed by De Vos et al. (1985) also presents problems

[7.5]

as the only characteristics presented by these authors to differentiate C. terrigena from P. testosteroni are utilization of carbon sources. It has since been proposed that P. acidovorans and P. testosteroni should be transferred to the genus Comamonas (Tamaoka, Ha & Komagata, 1987) but we disregard this as it is difficult to distinguish the two genera with conventional tests. Minidefinition: Pseudomonas (alkali-producers): Gram-negative rods; motile by polar flagella. Aerobic. Catalase-positive; oxidase-positive. Produce alkali in the open tube of O-F medium; attack few or no carbohydrates. 7.6

The actively saccharolytic motile rods (Achromobacter, Agrobacterium, Janthinobacterium, oxidizing pseudomonads, Flavobacterium, Weeksella). Characters common to members of the group: Gram-negative rods. Strict aerobes. Catalase-positive; oxidase-positive. Produce acid from many sugars in ammonium salt medium. Motile (except Pseudomonas mallei).

Bacteria that produce pigmented colonies are easy to recognize but they can cause difficulties in biochemical tests (e. g. oxidase) that involve a colour reaction. In such instances, non-pigmented variants, if produced, can be used instead. For other organisms, media have been devised to encourage pigment production and Mannitol Yeast-extract Agar (Appendix A2.4.2) is especially useful for Janthinobacterium and Chromobacterium species. The temperature of incubation is at least as important as the medium and stronger pigments are usually produced at the lower end of the growth range; 20-25 C is satisfactory for pigment production by most mesophilic organisms. The group is heterogeneous but all species come within the descriptive term 'actively saccharolytic'; they invariably show acid production from glucose in suitable media. All the species oxidize carbohydrates and this is best seen in basal media with a low peptone content such as Ammonium Salt (Appendix A2.5.5) and O-F sugars or in buffered single substrate (BSS) tests (Pickett & Pedersen, 1970*). 109

Table 7.6a. Second stage table for Ochrobactrum, Achromobacter, Agrobacterium, Chryseomonas, Flavimonas, Janthinobacterium and Pseudomonas l Motihty Growth at 37 °C Growth at 20-22 °C Brown pigment Violet pigment Green pigment Yellow pigment Orange pigment Growth on MacConkey Agar Oxidase Alkaline in O-F medium Nitrate reduced to nitrite Simmons' citrate medium Christensen's citrate medium Urease Gelatinase production Growth in KCN medium H2S (PbAc paper) Gluconate Malonate ONPG Carbohydrates acid from: 10% glucose 10% lactose [in Peptone Water Medium], acid from: glucose [in ammonium salt media], acid from: glucose adonitol arabinose cellobiose ethanol fructose glycerol inositol lactose maltose mannitol raffinose rhamnose salicin sorbitol sucrose trehalose xylose Phenylalanine Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Selenite reduction Casein hydrolysis Deoxyribonuclease production Thornley's arginine Tween 20 hydrolysis Tween 80 hydrolysis Tyrosine hydrolysis Tyrosine, brown pigment from Nitrite reduced PHBA* growth PHBA* accumulation in cells Aesculin hydrolysis Growth in presence of cetrimide Fluorescence - King's medium B Growth at 5 °C Growth at 42 °C 3-ketolactose production Lecithinase production Starch hydrolysis

+

+

+

d

16

17

18

19

+

d

d

+

+

+

d

+ +

d -

d -

-

-

-

-

+

-

-

-

d

-

+

d

+ -

+ -

+ +

+ +

+ +

+ +

+ d

-

+ +

+ +

+ +

+

d +

A U

A U

-

_ d

_ -

_ +

_

_ d

_ d

__ +

A U

A U

d d"

-

d

+

-

+

d

+

+ d + + -

+ d d -

-

-

+ +

+ +

+ +

d d

d

+

A U

+ _ d

d

d +

+

-

-

d _

d _

d + d d +

d d d _ d d

d d d — d -

d d -

+ d d — d —

-

d

_ +

_ _

-

_

+

d -

d -

—

_

_

+ +

+ +

-

+ d

+ +

d d

d

_

—

+ +

+ +

-

-

-

-

-

d

-

-

-

d

d

d

-

-

-

+ + + +

+ + +

+ -

+ + + +

+ + +

+ + -

+ + -

+ d + +

+ + -

+ + + +

+ d + d

+ + d

d -

+ + +

+

d d -

+ + + + + + + + + + + d +

_ d d d + d + + + +

+

+

+ + + _ + + + d +

+ + + + + + + d +

_ + + + + + + d + + d +

d + d d d d d d d

_ d + + d d d +

_ d + -

+ + d + + + d d + +

+ + + + + + + + + +

+

+ + + _ + + d + + -

+ + + _ d

d d + + d + d d -

+ d + + + d -

d d + _ + + -

d + + d + + + _ + -

+ d + + + d + d + + d +

d + d + + -

d + d + + + + -

+ d + d + + + + d + + d + d +

da d" + + + + + + d d d

+ _ d + d d + d

+ _ + + d + -

d _ + + + + + +

+ + d d -

+ + _ d

d _ d d d

-

-

d

+

+

d

d

d

1 Ochrobactrum anthropi; Achromobacter Group A; Achromobacter species biotypes 1 and 2; Groups Vd-1 and Vd-2 2 Achromobacter Group B 3 Achromobacter xylosoxidans; Alcaligenes xylosoxidans subspecies xylosoxidans 4 Agrobacterium tumefaciens; Agrobacterium radiobacter 5 Agrobacterium Yellow Group 6 Chryseomonas luteola; Group Ve, type 1 7 Flavimonas oryzihabitans; Group Ve, type 2 8 Janthinobacterium lividum; 'Chromobacterium lividum' 9 Pseudomonasaeruginosa; 'P.pyocyanea' 10 Pseudomonas cepacia; 'P. multivorans'; 'P.kingii' *PHBA: Poly-P-hydroxybutyrate. a More strains positive by Richard's (1968) method. b Cannot use nitrate as N source. Other symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

d -

d

— d -

-

+ d

d -

-

+

d

-

-

+ + +

+ d + +

+ — + -

+ -

+ d d

_ + + + + d d

d + + + + + + _ + d +

d d d _ _ _ -

d + d d d -

+ + d -

d

A U

+

d + + — d -

_ -

+

d + _ + +

-

+

d

d d _

+ + + + + + d d + d

d -

+

15

+

-

+

+

14

— +

+

d +

+

13

— -

+

+

+

12

+

+

+

+

11

+

+

d +

d

10

d + + + d + + + -

+ + + _ + + d

+ + + + d + — + + d d +

+ + d + d

d

d -

+ -

-

+ _ + d d -

d d + + d d + +

+ d d +

d d +

+ + -

d d

-

11 Pseudomonas fluorescens 12 Pseudomonas mallei; 'Loefflerella mallei'; 'Pfeifferella mallei'; 'Malleomyces mallei' 13 Pseudomonas maltophilia; Xanthomonas maltophilia 14 Pseudomonas paucimobilis; 'Sphingomonas paucimobilis' 15 Pseudomonas pickettii 16 Pseudomonas pseudomallei; 'Loefflerellapseudomallei'; 'L whitmor(e)i'; 'Pfeifferella whitmori'; 'Malleomyces pseudomallei' 17 Pseudomonas putida 18 Pseudomonas stutzeri 19 Pseudomonas vesicularis

CHARACTERS OF GRAM-NEGATIVE BACTERIA

7.6.1 Achromobacter (Tables 7.5; 7.6a). This was for many years an ill-defined genus of motile Gram-negative rods whose main distinguishing feature was the negative one of not producing pigmented colonies. The genus was never adequately defined and Achromobacter became the name of the dump-heap of organisms left unnamed by the rejection of the generic name 'Bacterium' by the Judicial Commission (Opinion 4 (revised), 1954). Accord-ingly, it was omitted from the Approved Lists of Bacterial Names (1980) but was later revived by Yabuuchi & Yano (1981) for the single species Achromobacter xylosoxidans, which is of clinical interest (Holmes, Snell & Lapage, 1977). Since this organism is closely related to Alcaligenes denitrificans, Kiredjian et al. (1986) proposed that Achromobacter xylosoxidans should be transferred to the genus Alcaligenes with two subspecies, Alcaligenes xylosoxidans subsp. xylosoxidans and Alcaligenes xylosoxidans subsp. denitrificans. Despite good scientific evidence for this proposal we prefer to keep Achromobacter xylosoxidans and Alcaligenes denitrificans as separate taxa (see Section 7.5.1). The characteristics of Achromobacter xylosoxidans are compared with Alcaligenes in Table 7.5 and with the actively saccharolytic non-fermenters in Table 7.6a. In a numerical taxonomic analysis of Achromobacter-likt strains from clinical material Holmes & Dawson (1983) recognized six groups, A to F, of which A and B were by far the largest. Recent taxonomic work shows that groups A, C and D are biotypes of a single species which is not phylogenetically closely related to Achromobacter xylosoxidans; the name Ochrobactrum anthropi has been proposed for this species. Groups B, E and F still require further taxonomic investigation but we suspect that group B will also ultimately reside in a new genus created to accommodate it. We show Achromobacter group B in Table 1.6a together with Ochrobactrum anthropi biotype A (= Achromobacter group A, which also includes organisms formerly described either as Achromobacter biotypes 1 and 2 or as Groups Vd-1 and Vd-2 of Clark et al, 1984). Minidefinition: Achromobacter. Gram-negative rods; motile (by peritrichous flagella). Aerobic. Catalase-positive; oxidase-positive. Attack sugars by oxidation. Type species: A. xylosoxidans; NCTC strain 10807. 112

[7.6]

7.6.2 Agrobacterium (Table 7.6a). This genus comprises primarily pathogens of plants; indeed, the many species within it have been defined by plant pathologists largely according to their pathogenic effects on plants. Several studies show that a different classification can be achieved when the phytopathogenic effect of strains is not given undue weight. Kersters & DeLey (1984a) recognized three species: A. tumefaciens (synonym of A. radiobacter), A. rhizogenes and A. rubi. In addition Holmes & Roberts (1981) described a fourth taxon, the 'Agrobacterium yellow group', which produced yellow-pigmented colonies and were included in Agrobacterium because they formed 3-ketolactose, a test considered specific for agrobacteria (Bernaerts & DeLey, 1963). The 'Agrobacterium yellow group' is, however, probably much more closely related to Pseudomonas paucimobilis as the two differ little in conventional biochemical characters (Table 7.6a). Strains of the group have been isolated from dialysis fluid (Swann et al., 1985) and A. tumefaciens and other agrobacteria occasionally from other clinical specimens (Lautrop, 1967; Riley & Weaver, 1977; Kiredjian, 1979; Plotkin, 1980; Freney et al, 1985). Minidefinition: Agrobacterium. Gram-negative rods; motile. Aerobic. Catalase-positive; oxidase-positive. Wide range of sugars attacked by oxidation. Type species: A. tumefaciens; ATCC strain 23308. 7.6.3 Janthinobacterium (Tables 7.6a; 7.8a). Janthinobacterium lividum formerly resided in the genus Chromobacterium together with C. violaceum (DeLey, Seagers & Gillis, 1978). Both these organisms are characterized by the production of violet pigments, which make determination of the oxidase reaction difficult with methods that depend on the production of a purple colour in the test. Snell (1973) was able to find non-pigmented variants suitable for the oxidase test; in their absence, very young cultures are less pigmented and should be used. To encourage pigment production we recommend the use of Mannitol Yeast-extract Agar incubated at 20-25 C (20 °C for Janthinobacterium lividum and at 25 °C for C. violaceum). We deal with Chromobacterium violaceum in Section 7.8.4. Chromobacterium was a genus created around a colour and it seems to be as unsuccessful as other

ACTIVELY SACCHAROLYTIC RODS

genera confined to a particular colour. J. lividum and C. violaceum are both Gram-negative rods but there are several important differences between them: J. lividum grows between 4° and 30 °C, C. violaceum between 10° and 37 °C. J. lividum oxidizes carbohydrates and its characters are shown in Table 1.6a; most strains of C. violaceum ferment sugars (a few do so oxidatively) and it is shown with the fermenting Gram-negative rods in Table l.Sa together with the characters of J. lividum beside it for comparison. At one time or another both species have been labelled Chromobacterium violaceum, and in literature before 1958 the names should be viewed with caution. Chromobacteriosis is a rare disease of man caused by the mesophilic species Chromobacterium violaceum (Sneath, 1960); the psychrophile is unlikely to be isolated from clinical material but may arise as an infrequent contaminant of stored refrigerated products such as blood. Mini definition: Janthinobacterium. Gramnegative rods; motile. Produce a violet pigment. Aerobic; psychrophilic. Catalase-positive; oxidase-positive. Sugars attacked by oxidation. Type species: J. lividum; NCTC strain 9796. 7.6.4 Pseudomonas: the oxidizers (Table 7.6a). This large group of organisms includes pathogens for both animals and plants, but because specimens from animal and plant tissues are studied in different laboratories different tests and techniques are often used; this has resulted, in the past, in a separation into animal and plant species which is quite unjustified, for strains pathogenic for plants may also be pathogenic for animals and vice versa. Indeed, different names may be proposed for the same organism by the different groups of workers. For example, P. cepacia was proposed for an organism isolated from onions, and later 'P. multivorans" was suggested for the same organism isolated from soil and clinical material from man (see Holmes, 1986£). Until recently, Pseudomonas aeruginosa (previously referred to as 'Pseudomonas pyocyaned* in medical laboratories) and P. fluorescens were virtually the only species studied extensively by both groups of workers, but the common ground has now been much extended. Pseudomonas aeruginosa produces two water-sol-

[7.6] uble pigments: pyocyanin which gives the green-blue appearance to the areas surrounding colonies or confluent growth on plates; and fluorescin, yellow in colour but which, as its name implies, exhibits characteristic fluorescence in ultra-violet light. While pyocyanin is formed only by P. aeruginosa, another green pigment, chlororaphin, is produced by some plant pseudomonads; this pigment is not water-soluble and is seen as crystals in agar media. Other pseudomonads, such as P. putida, may produce a fluorescent pigment similar to fluorescin. Although not closely related phylogenetically, P. pseudomallei, originally known as Whitmore's bacillus, has much in common with the fluorescent pseudomonads. Wetmore & Gochenour (1956) found that growth of this organism was generally inhibited on media such as Salmonella-Shigella (SS) Agar, Deoxycholate Citrate Agar, and Cetrimide Agar, on which the fluorescent species would grow. The flagella of P. pseudomallei were first described as peritrichate by Legroux & Genevray (1933), but later as polar by Brindle & Cowan (1951) and by Wetmore & Gochenour (1956); all these workers showed that the organism had much in common with the pseudomonads but none suggested transferring it to that genus and it was not included in Pseudomonas until the seventh edition of Bergey's Manual (Haynes, 1957). Redfearn, Palleroni & Stanier (1966) included P. mallei (the glanders bacillus) in their survey of Pseudomonas species, and regarded it as a typical but permanently non-motile member of that genus. This organism is closely related phylogenetically to P. pseudomallei. Fortunately, glanders is almost a disease of the past and few now have the opportunity (or the risks) of working with recently isolated cultures. Melioidosis (of which P. pseudomallei is the causative organism) is a disease of tropical countries but occasionally occurs in temperate climates in people who were probably infected abroad (Peck, 1986). Pseudomonas pseudomallei may be confused with other pseudomonads and although its wrinkled colonial appearance may be characteristic, it is not diagnostic as it can be simulated by P. aeruginosa and by P. stutzeri (Lapage, Hill & Reeve, 1968). Zierdt & Marsh (1971) state that after incubation for three or four days, colonies of P. pseudomallei have an aromatic odour that is distinctive; but without experience 113

CHARACTERS OF GRAM-NEGATIVE BACTERIA

of it we do not think that this subjective characteristic can be of much help for identification. Other diagnostic features are the development of bright red colonies on MacConkey agar (and on the basal medium without lactose) and the ability to grow on Deoxycholate Citrate Agar. For most laboratories the isolation of Pseudomonas pseudomallei is a rare event and confirmation of isolates should always be obtained from a reference laboratory with access to specific antiserum tested against Pseudomonas aeruginosa and other pseudomonads to exclude cross-reactions. P. mallei is one of the most dangerous organisms to work with in a laboratory, and P. pseudomallei, though probably less dangerous, should be treated with the same respect; both are listed in Hazard Group 3 by the Advisory Committee on Dangerous Pathogens (1984). Many other pseudomonads have been isolated from clinical material and the hospital environment (see, for example, Gilardi, 1978, 1985a,Z?) and there has been much effort to find distinguishing features to assess the potential pathogenicity of the different species. Undoubtedly some strains are 'opportunist pathogens' and, as many of them are resistant to disinfectant solutions, they may cause low-grade infections from contaminated catheters, wash-out fluids, and drip lines. Media for the selective isolation of pseudomonads have been reviewed by Park & Billing (1965). Thorn et al. (1971) found that the addition of nitrofurantoin to King's B medium (Appendix A2.4.3) was easier to prepare than cetrimide media and was equally good for the selective isolation of Pseudomonas aeruginosa. On this medium most strains of Proteus species, Escherichia coli, Enterococcus faecalis, and staphylococci were either inhibited completely or grew only feebly after overnight incubation at 37 C. Tests to identify the many species are becoming more complex; for example, one of the distinguishing characters is to show by phase-contrast microscopy the presence or absence of poly-p-hydroxybutyrate (PHB) in the cells of cultures grown preferably in a medium with DL-p-hydroxybutyrate as the carbon source, with subsequent confirmation by staining with Sudan Black B. Apart from the accumulation of PHB in the bacterial cells, the tests for characters shown in Table 7,6a do not call for exceptional knowledge or skill and can be performed in most lab114

[7.6]

oratories. The growth of P. aeruginosa is often accompanied by iridescence; the mechanism of this phenomenon is still unknown, but it is independent of phage sensitivity (Zierdt, 1971). Four methods are available for typing strains of P. aeruginosa for epidemiological purposes: bacteriocin typing, serotyping by somatic and by flagellar antigens, and bacteriophage typing (Pitt, 1988). Bacteriocin typing is probably the most popular and since the original description of Gillies & Govan (1966) the technique has been modified to increase its sensitivity and specificity (Fyfe, Harris & Govan, 1984). The discrimination achieved by pyocin typing between clinical isolates of P. aeruginosa is adequate for most investigations but the test reproducibility is influenced by media composition, bacterial inoculum and storage of the reference indicator strains. Pseudomonas aeruginosa is heterogeneous in Osomatic antigen structure and 17 heat-stable (lipopolysaccharide) antigens form the basis of the International Antigenic Typing Scheme (I ATS) described by Liu et al. (1983). O-typing by slide agglutination is reproducible and the number of clinical strains typable is high (90-95%); few strains give unstable suspensions in saline but about 5% of isolates are polyagglutinable. The frequency of serologically atypical isolates increases greatly in chronic infections, especially cystic fibrosis (Pitt et al., 1986). The discriminatory power of O-typing is fair but certain groups such as O6, O10, and O i l predominate and together account for over 40% of all strains. Subtyping by minor antigens is useful in dividing strains of serogroup O6 (Pitt, 1980a). About 90% of clinical strains are typable with flagella (H) antisera. In combination with O-typing, H antigens increase discrimination between strains as many combinations of H factors occur (Lanyi & Bergan, 1978). Some H antigens undergo diphasic variation and this property adversely affects the reproducibility of H-typing (Pitt, 1980Z?). Bacteriophage typing has been used extensively by various workers and a standard set of phages is recommended (Bergan, 1978). Some laboratories favour the coding of lytic results by designating distinct patterns with numbers but others prefer to compare the patterns after taking account of the known variability of the method. Phage-typing cannot be used alone for precise type-identification despite the

[7.6]

ACTIVELY SACCHAROLYTIC RODS

Table 7.6b. Buffered single substrate (BSS) tests for Pseudomonas( Pickett & Pedersen, 1970b,c)

Acid from: glucose fructose lactose maltose mannitol salicin sucrose xylose

+ + — + — +

+ + + + + + d +

-

+ +

—

+ +

d

? ?

+ +

d -

+ +

+

+ d

+ +

+ +

— + + +

d

d -

+ +

-

—

+

+ d -

— +

+

1 2 3 4 5 6

Pseudomonas aeruginosa; 'P. pyocyanea' Pseudomonas cepacia; 'P. multivorans'; 'P. kingii' Pseudomonas diminuta Pseudomonas fluorescens Pseudomonas maltophilia; Xanthomonas maltophilia Pseudomonas pseudomallei; 'Loefflerella pseudomallei', 'L. whitmor(e)V; 'Pfeifferella whitmori', 'Malleomyces pseudomallei' 7 Pseudomonas putida 8 Pseudomonas stutzeri Symbols used in the Table are explained in Tables 5.1 and 5.2 on p.47.

high typability as there is considerable overlapping of patterns and lysis by some phages is particularly frequent. It is nevertheless a useful adjunct to O-serological typing. The acid production from carbohydrates shown in Table 7.6a occurs when the organisms are grown in media without peptone (ASS, Appendix A2.5.5). Media containing only a small amount of peptone (e.g. Hugh & Leifson base, Appendix A2.5.1) may also be used. Acid may be produced from more carbohydrates in the buffered single substrate tests of Pickett & Pedersen (1970&, c) in which a heavy suspension of the test organism is added to the carbohydrate+buffer+indicator+bacteriostatic-agent mixture. The differences between the results of the two kinds of test can be seen by comparing Tables 1.6a andb. Pseudomonas aeruginosa produces alkali on Christensen's urea medium and, more slowly, on the base without urea. Many pseudomonads break down arginine but M0ller's method is not always satisfactory with these organisms; Sherris et al. (1959) and Thornley (1960) have described suitable methods. For detecting lysine and ornithine decarboxylases of pseudomonads, Richard's (1968) methods are more sensitive than those of M0ller (Snell et al, 1972).

Nutritional requirements can also provide diagnostic characters for some pseudomonads: thus Pseudomonas maltophilia requires methionine, and P. diminuta (Section 7.5.3.) needs cysteine for growth. Pseudomonas maltophilia, originally described by Hugh & Ryschenkow (1961) as an Alcaligenes-\\ke species, is unusual in producing acid from maltose in Hugh and Leifson sugars before it is seen in glucose, which often remains negative in ASS media. Park (1967) found that the glucose was attacked even though acid was not produced. P. maltophilia is also unusual for a pseudomonad in that different strains give different results in the oxidase test. Hugh & Ryschenkow found that 10 of 26 strains gave positive results and Holmes, Lapage & Easterling (1979) likewise found 25 of 117 strains positive, but Pickett & Pedersen (1970b) had only 3 positive reactions among their 27 strains; the variability, apart from the actual strains, may be due to differences in sensitivity of the reagents used for the test (Snell, 1973). Species described more recently include P. pickettil (Ralston, Palleroni & Doudoroff, 1973) and P. paucimobilis (Holmes et al, 1977), comprising strains from clinical material and hospital equipment. The latter organism and the so-called 'Agrobacterium yellow group' (7.6.2.) are closely related. P. men115

CHARACTERS OF GRAM-NEGATIVE BACTERIA

docina closely resembles P. stutzeri but we do not include it in Table 1.6a as it is thought to occur only rarely in clinical material; the characteristics for differentiating them are given by Holmes (1986a). P. vesicularis is phylogenetically closely related to P. diminuta (Table 7.5) but it differs from the latter organism in being orange-pigmented and in producing acid from a wider range of carbohydrates. Also included in Table 1.6a are the yellow-pigmented oxidase-negative organisms of 'Group Ve-1' and 'Group Ve-2' of Clark et al. (1984): although generally regarded as Pseudomonas-like with the names Pseudomonas luteola and Pseudomonas oryzihabitans proposed for them (Kodama, Kimura & Komogata, 1985), they are now named respectively Chryseomonas luteola and Flavimonas oryzihabitans (Holmes etaL, 1987). Minidefinition: Pseudomonas (oxidizers). Gram-negative rods; motile (by polar flagella). Aerobic. Catalase-positive; oxidase-positive. Attack sugars by oxidation. Fluorescent, diffusible yellow pigment may be produced by some species. Type species: P. aeruginosa; NCTC strain 10332. 7.6.5 Flavobacterium (Table 7.6c). Flavobacterium has long been regarded as an unsatisfactory genus, made up of a heterogeneous collection of species which formed yellow colonies. Although the type species is no longer regarded as a flavobacterium, the genus has been re-defined (Holmes, Owen & McMeekin, 1984a) and now comprises seven welldefined taxa that fall into three natural groups which are rather different to the groups previously described by Pickett & Pedersen (1970a,Z?,c). The first group comprises three saccharolytic, indole-positive species; the second a single non-saccharolytic indolenegative species; and the third group three saccharolytic, indole-negative species. As the last three species contain novel sphingophospholipids in their cell walls, a new genus Sphingobacterium has been proposed for them (Yabuuchi et al, 1983). We feel, however, that this characteristic alone is insufficient to warrant a new genus and so we retain these three species in Flavobacterium. The three taxa comprising the first natural group are not easily differentiated from each other and one of them, Flavobacterium species Group lib, is heterogeneous in conventional 116

[7.6]

biochemical tests. The name F. indologenes has been proposed for the whole of Group lib but it is also heterogeneous by DNA-DNA hybridization; the name F. gleum has been proposed for certain strains of Group lib showing a high degree of relatedness (Holmes et al., 1984&). Until this nomenclatural problem is resolved we prefer to use the original designation Flavobacterium species Group lib. F. meningosepticum is the species of major clinical interest (Holmes, 1987). Colonies of Enterobacter sakazakii and Erwinia herbicola (known in the USA as Enterobacter agglomerans) characteristically produce yellow colonies. In other fields some species of Pseudomonas and Xanthomonas produce yellow pigments, and neglect to determine the Gram reaction of isolates may cause some difficulty with a few Grampositive species. Indole production by those species that do so is weak and for demonstration of this character extraction with xylene followed by the addition of Ehrlich's reagent is recommended. The indole-producing but non-pigment-forming taxa, Groups Ilf and IIj, often regarded as Flavobacterium - like organisms which could have formed a fourth natural group in that genus, have been transferred recently to a new genus, Weeksella, by Holmes et al. (1986a,&). We deal with the species in this genus in Section 7.6.6 but it should be noted that they do not attack carbohydrates. Minidefinition: Flavobacterium. Gramnegative rods; non-motile. Aerobic. Catalase-positive; oxidase-positive. Colonies yellow-pigmented. Carbohydrates attacked slowly by oxidation or not at all. Resistant to most antimicrobial agents. Type species: F. aquatile; ATCC 11947. 7.6.6 Weeksella (Table 7.6c). This new genus was proposed by Holmes et al. (1986a,fr) to accommodate the two indole-producing, non-saccharolytic Flavobacterium-like taxa, Group Ilf, isolated mostly from the female genital tract (Mardy & Holmes, 1988), and Group IIj, commonly associated with animal bites (Rubin, Granato & Wasilauskas, 1985). Unlike Flavobacterium taxa, however, they do not form yellow-pigmented colonies and they are highly susceptible to most antimicrobial agents. The name

[7.6]

ACTIVELY SACCHAROLYTIC RODS

Table 7.6c. Second-stage table for Flavobactenum and Weeksella 6

7

8

9

Carbohydrates [in ammonium salt media], acid from: glucose adonitol arabinose cellobiose ethanol glycerol lactose maltose mannitol raffinose rhamnose salicin sucrose trehalose xylose Aesculin hydrolysis Casein hydrolysis Gelatinase production Growth at 42 °C Indole production (Ehrlich reagent) Nitrate reduced to nitrite Nitrite reduced ONPG Starch hydrolysis Urease 1 2 3 4 5 6 7 8 9

Flavobactenum breve Flavobactenum meningosepticum Flavobactenum species Group lib; F. gleum; F. indologenes Flavobactenum odoratum; Group M-4f Flavobactenum multivorum; Group IIk-2; Sphingobacterium multivorum Flavobactenum spiritivorum; Sphingobacterium spiritivorum Flavobactenum thalpophilum Weeksella virosa; Group Ilf Weeksella zoohelcum; Group IIj

Symbols used in the Table are explained in Tables 5.1 and 5.2 on p.47.

Weeksella virosa has been proposed for Group Ilf and W. zoohelcum for Group IIj organisms (Holmes et al, \9&6a,b). W. virosa produces extremely mucoid colonies and, unlike W. zoohelcum, grows on MacConkey agar. Minidefinition: Weeksella. Gram-negative rods; non-motile. Aerobic. Catalase-positive; oxidase-positive. Do not produce yellow-pigmented colonies. Carbohydrates not attacked. Sensitive to most antimicrobial agents. Type species: W. virosa; NCTC strain 11634.

7 .7

The Pasteurella and pasteurella-like group (Pasteurella, Actinobacillus, Cardiobacterium, Francisella) Characters common to members of the group: Gram-negative rods; non-motile at 37 °C. Aerobic and facultatively anaerobic. Oxidase-positive. Attack sugars fermentatively.

This group of organisms is still heterogeneous and relatively uncommon as pathogens of man despite the transfer of the plague organism and its close relations 117

CHARACTERS OF GRAM-NEGATIVE BACTERIA

to the genus Yersiniu. However, when they do cause infection, they pose a challenge to both microbiologist and clinician in terms of diagnosis and treatment. We describe the Yersinia species in Section 7.9.20 and give their characters in Table 7.9g but show them also in Table 7.7 for comparison with Pasteurella. Actinobacillus and Pasteurella have much in common and suggestions have been made for their fusion. Cardiobacterium was named in 1964 by Slotnick & Dougherty as an agent causing endocarditis (Wormser & Bottone, 1983) though it was first described as a Pasteurella-like organism by Tucker et al. (1962). Francisella, unlike the other genera in this group, requires specially enriched media for isolation (Berdal & S0derlund, 1977). The original species of this genus, previously regarded as Brucella-like or as Yersinia-MkQ, was placed in the genus Pasteurella in the 7th edition of Bergey's Manual (1957). Following taxonomic work (Owen, 1974), it was placed in a new genus and renamed Francisella tularensis to commemorate the pioneering work of Edward Francis on tularaemia. The genetic identity of this genus and its subdivision have since been confirmed (Olsufiev & Meshcherykova, 1983). In Table 7.7 we include two organisms, Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus, which have much in common with each other. They are both small Gram-negative coccobacilli which grow in broth as 'granules' adherent to the tubes; on plates, colonies of A. actinomycetemcomitans stick to the medium. Growth of both is improved by CO2; and neither of them needs X or V factors although H. aphrophilus has an apparent X factor requirement on first isolation. The latter is probably placed correctly in the genus Haemophilus where we discuss it more fully. Recent taxonomic work (Potts, Zambon & Genco, 1985) suggests that A. actinomycetemcomitans could also be transferred to Haemophilus though its lack of X or V factor requirements would conflict with this genus as currently defined. We therefore show both these organisms in Table 7.7 as well as with Haemophilus in Table 7.10/7. Lastly, as before, we again include Aeromonas salmonicida, previously called Necromonas, in Table 7.7 as its characters seem to fit quite well in this group. Although it has no known medical signifi118

[7.7]

cance, it can be isolated from fish and, despite the validity of its current name, we suspect that it will not remain in the genus Aeromonas for ever. 7.7.1 Pasteurella (Table 7.7). Several species have been described for this genus, which at one time also included the 'plague bacillus' - now named Yersinia pestis - together with Y. pseudotuberculosis and Y. enterocolitica. Pasteurella multocida represents an 'umbrella' species which includes numerous strains that were originally named after the animals from which they had been isolated; sensibly, however, the single epithet multocida was later applied to the commoner strains which caused haemorrhagic septicaemia and abscesses in animals of all kinds, including man. Cutaneous infections in man also occur following animal scratches and bites, particularly from cats and dogs. Subsequently, subdivisions of this unified species were again made and these included P. haemolytica (Newsom & Cross, 1932) and P. pneumotropica (Jawetz, 1950; Henriksen, 1962) as well as P. haemolytica var. ureae (Henriksen & Jyssum, 1960) which was later elevated to species rank as Pasteurella ureae (Jones, 1962; Smith & Thai, 1965). The biochemical characters of these species are shown in Table 7.7. The principles of the CAMP test (Appendix C3.7) were used by Bouley (1965) to differentiate between P. multocida (negative) and P. haemolytica (positive). Dye sensitivity tests, included in the second edition of this Manual, were also used previously not only to distinguish Pasteurella from Yersinia but also to differentiate between Pasteurella species (Midgley, 1966). As they are not often used for these purposes and now have little taxonomic value, we do not tabulate the test results but mention that thionin (1: 25 000), malachite green (1:50 000) and safranin O (1:25 000) in media should inhibit the growth of Pasteurella but not of Yersinia species. P. haemolytica was divided into serotypes by Biberstein, Gills & Knight, (1960) and into biotypes A (arabinose fermenters) and T (trehalose fermenters) by G.R. Smith (1961); the latter biotype is also catalase-negative (Biberstein & Gills, 1962). Freshly isolated strains of P. haemolytica produce clear zones of p-haemolysis on Blood Agar but this haemolytic ability may be reduced after several subcultures. Several serological schemes have been proposed

[7.7]

PASTEURELLA AND PASTEURELLA-LIKE GROUP

Table 7.7. Second-stage table for Pasteurella, Actinobacillus, Cardiobacterium, Yersinia and Francisella 1

2

3

4

5

6

7

w

8

9

10

11

12

13

14

15

16

17

Motility at 22 °C Oxidase Growth on MacConkey Agar Growth improved by CO2 Growth in KCN Carbohydrates, acid from: arabinose lactose mannitol raffinose salicin sorbitol sucrose trehalose xylose ONPG Aesculin hydrolysis

d 7 +

+ 7 -

+ 7 -

+ + 7 d

+ + 7 -

+ 7 -

+ -

d d

d (d)

-

d d

-

d

d (+)

_ + + + d + -

+

+ + _ + + + d d

+ + + + + + -

+

+

_ + + d d -

d + + + -

+

_

7

A U

-

d + d d d -

d + -

+ + -

7 + + 7

_ + 7

_ + 7

+ + 7

+ +

-

+ +

+ -

-

-

+

+ + + + + +

_ + + + + + +

+

_

-

+

7 + 7 7

d _ d -

9

7 _ + + -

7 7 + + 7

+ + -

1 2 3 4 5 6 7 8

d + d +

_ + + d

_ -

+ -

-

-

-

-

Pasteurella multocida; 'P. septica' Pasteurella pneumotropica Pasteurella uresae; 'P. haemolytica var. ureae' Pasteurella haemolytica type A; P. haemolytica Pasteurella haemolytica type T; P. trehalosi Pasteurella gallinarum Actinobacillus lignieresii Actinobacillus equuli; 'Shigella equuli'; 'S. equirulis'; 'Bacterium viscosum-equi'; 'B. nephritidis-equi' 9 Actinobacillus suis

a

_

_

d

(+) (+) -

10 11 12 13 14 15 16 17 18 19

+ + -

(+) + + -

19

-

d w -

7

_ + 7 -

_ + 7 -

_ + 7

+ 7 7 -

-

-

+ -

+ -

+ (d)

+ -

d 7 + + -

+ + + + + +

+ -

+ -

+

-

_ _ + + + -

?

+

_

+

7 + + -

7 -

7

_ + -

+ + + +

+ + +

+ + + +

i

Nitrite reduced Indole Gelatin hydrolysis Urease H2S Ornithine decarboxylase

18

7 -

7 + -

+

+ (+) + " 7

d +

-

+ d +

_ _ + -

A U

7 7 7 d 7 7 7 7 7 7 _a

7 + 7

Actinobacillus from sow's vagina (Ross et al. 1972); A. rossi 'Actinobacillus' actinomycetemcomitans Haemophilus aphrophilus Haemophilus paraphrophilus Cardiobacterium hominis Yersinia pestis; 'Pasteurella pestis'; plague bacillus Yersinia pseudotuberculosis; 'Pasteurella pseudotuberculosis' Yersinia enterocolitica; Pasteurella X Aeromonas salmonicida; 'Necromonas salmonicida' Francisella spp.

F. philomiragia often positive

Other symbols used in the Table are explained in Tables 5.1 and 5.2 on p.47.

for the subdivision of P. multocida (Steel, 1963); and Frederiksen (1971) divided this species biochemically into six and P. pneumotropica into three biotypes. There is a close relation between P. multocida and P. pneumotropica and it seems probable that changes in the taxonomic status of the latter may follow from current DNA hybridization work. P. ureae was isolated originally from respiratory infections in man. A new species, P. aerogenes, was proposed by McAllister & Carter (1974) for a group of organisms that produced gas from sugars and which were isolated from human infections following pig bites. These

organisms are non-haemolytic, indole-negative and urease-positive. Minidefinition: Pasteurella. Gram-negative rods. Non-motile. Aerobic and facultatively anaerobic. Usually catalase-positive; oxidase-positive. Sugars are attacked by fermentation. Type species: P. multocida; NCTC strain 10322. 7.7.2 Actinobacillus (Tables 7.7; 7.10£) consists of species that seem to have no obvious home. Organisms of at least two kinds are represented: the 119

CHARACTERS OF GRAM-NEGATIVE BACTERIA

actinobacilli proper based on the type species Actinobacillus lignieresii and another organism, A. actinomycetemcomitans, usually but not always associated with Actinomyces israelii in material from patients with actinomycosis. Cowan suggested that the latter organism, A. actinomycetemcomitans, was only placed in this genus by Topley & Wilson (1929) in the first edition of their book Principles of Bacteriology and Immunity because they could not think where else to put it, and it has since remained there. It closely resembles Haemophilus aphrophilus and would be more satisfactorily placed in that genus if the definition would allow it, but as it is not satisfactory in Actinobacillus we show it also in Table 1.10b for comparison with Haemophilus. A. lignieresii, found in cattle and sheep, is very similar to A. equuli and Wetmore et al. (1963) proposed that they should be amalgamated. A. equuli causes joint-ill and nephritis in foals. Colonies of both species are sticky and difficult to remove from the surface of agar media. Both of these non-motile, urease positive species have been associated with human infections and can be differentiated by trehalose and melibiose fermentation. A. suis strains usually produce a clear haemolytic zone on Blood Agar and hydrolyse aesculin. Mini definition: Actinobacillus. Non-motile Gram-negative rods. Aerobic and facultatively anaerobic. Catalase-positive; oxidasepositive (except A. actinomycetemcomitans). Sugars are attacked fermentatively without gas production. Nitrate reduced. Type species: A. lignieresii; ATCC strain 4189. 7.7.3 Cardiobacterium (Table 7.7). This pleomorphic organism is Gram-negative but it may retain sufficient crystal violet stain to give the appearance of metachromatic granules (Slotnick & Dougherty, 1964); the bacilli also usually have swollen bulbous ends resembling 'teardrops', which form characteristic rosette clusters. Like Haemophilus, it prefers a high humidity for growth, so incubation in a jar is advisable. Carbon dioxide improves growth but is not essential though it may appear so on first isolation; growth under strictly anaerobic conditions is good. Only one species, C. hominis, has been identified: it is probably part of the normal flora of the nose and throat of man but may occasionally cause endocarditis in sus120

[7.7]

ceptible persons with cardiac lesions (Weaver, Tatum & Hollis, 1972; Wormser & Bottone, 1983). Minidefmition: Cardiobacterium. Non-motile, pleomorphic Gram-negative rods sometimes with Gram-positive traces. Aerobic in a humid atmosphere and facultatively anaerobic. Catalase negative; oxidase-positive. Sugars fermented. Type species: C. hominis; NCTC strain 10246. 7.7 .4 Francisella (Table 7.7) currently contains two species of medical importance, F. tularensis (McCoy & Chapin, 1912) and F. novicida (Larson, Wicht & Jellison, 1955). F. tularensis, the cause of the plague-like illness tularaemia in man, was previously placed in the genus Pasteurella. It contains at least two biotypes with differing geographical spread and pathogenicity (Olsufiev, Emelyanova & Dunaeva, 1959; Olsufiev, 1970; Eigelsbach & McGann, 1984); in addition, despite some phenotypic differences between F. tularensis and F. novicida, Hollis et al. (1989) regard the latter as a further biotype of F. tularensis. These organisms are transmitted to man from a variety of zoonoses in wild animals, including rabbits, beavers, squirrels and game birds, either through direct contact or by biting insects. For isolation and growth, which is relatively slow, F. tularensis, the most exacting, requires media enriched with blood, cysteine and glucose (Berdal & S0derlund, 1977; Eigelsbach & McGann, 1984). In fresh cultures, the organisms are small, non-motile, often capsulated and cocco-bacillary in form; in older cultures, they show marked pleomorphism. They are catalase-positive, oxidase-negative, produce H2S and form acid but not gas from glucose and several other sugars. The possession of a citrulline-ureidase system distinguishes virulent strains of F. tularensis from the less virulent (Marchette & Nicholes, 1961). These organisms must be handled with respect and precautions taken to avoid laboratory-acquired infections; indeed serological procedures such as slide agglutination reactions and fluorescent microscopy with specific antisera are usually used for identification. For these reasons, although we include the collective reactions for Francisella in Table 7.7, we do not recommend the use of conventional characterization tests for their identification in routine laboratories.

PASTEURELLA AND PASTEURELLA-LIKE GROUP The organism isolated from water and the muskrat by Jensen, Owen & Jellison (1969), previously included in Yersinia, is genetically similar to Francisella but is oxidase-positive and salt-tolerant. Hollis et al. (1989), who have isolated similar organisms from clinical infections in man, accordingly suggest that it should be transferred to Francisella as F. philomiragia. Minidefinition: Francisella. Gram-negative rods. Non-motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Sugars attacked fermentatively. Type species: F. tularensis; ATCC strain 6223. 1.1.5 Yersinia (Tables 7.7; 1.9g) is an offshoot of Pasteurella. It is now included among the enterobacteria and we deal with it in Section 1.9g. To show the similarities to and differences from Pasteurella, the characters of Yersinia are given also in Table 7.7. We repeat the Minidefinition here. Minidefinition: Yersinia. Gram-negative rods; motile or non-motile (temperature dependent). Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Sugars attacked fermentatively, occasionally with production of gas. Grow on MacConkey agar. Type species Y. pestis: NCTC strain 5923. 7.7.6 'Necromonas', a single-species genus created by I.W. Smith (1963) for the organism causing furunculosis of fish and previously included in this Manual, is no longer extant. This organism has now been transferred to the genus Aeromonas and we deal with it in Sections 7.8 and 7.8.2. 7.7.7 'Haemophilus' aphrophilus (Tables 7.7; 7.10£) seems to be X-factor-dependent on first isolation (Boyce, Frazer & Zinnemann, 1969) but loses this requirement rapidly on subculture. King & Tatum (1962) thought this species had much in common with Actinobacillus actinomycetemcomitans but doubted if it would be any better placed in that genus than in Haemophilus; however, Potts, Zambon & Genco (1985) suggest that they are both more akin to haemophili and should be transferred to that genus. For comparison of these two species we show

[7.7]

Haemophilus aphrophilus in Table 1.1a and the actinobacillus in Table 1.10b. 7.8

The Vibrio and vibrio-like group (Vibrio, Aeromonas, Plesiomonas, Chromobacterium violaceum) Characters common to members of the group: Gram-negative rods. Aerobic and facultatively anaerobic. Motile; catalasepositive; oxidase-positive. Attack sugars fermentatively. Nitrates are reduced by most species.

This group of organisms is fairly homogeneous so that, apart from the pigment produced by Chromobacterium violaceum, it is easier to cite characters in common than those by which the genera can be distinguished. Knowledge about the taxonomy, ecology and pathogenicity of this group has now increased considerably and, as forecast in the last edition of this Manual, the generic names Beneckea and Lucibacterium have been abolished and their species incorporated within Vibrio; the genus Photobacterium remains intact but we do not include it here as no pathogenic species are known. Several new Vibrio species have also been described, many of which are regarded as opportunist pathogens for man or aquatic animals. The microaerophilic species previously included in Vibrio and exemplified by 'V. fetus' have now been transferred to the genus Campy lobacter. Based on 5S rRNA sequencing, MacDonell & Col well (1985b) have suggested that Vibrio species pathogenic for fish should be placed in a new genus, Listonella, but as this awaits ratification, we continue to regard them as vibrios. The genus Allomonas, with a single species, A. enterica, proposed by Kalina et al. (1984) is probably identical with V.fluvialis. The taxonomy of Aeromonas still remains far from satisfactory. The case for placing the salmondisease organism in a separate genus, 'Necromonas\ has not been accepted although in contrast to the other Aeromonas species it is non-motile, grows at 5 °C but not at 37 °C, is fermentative and can produce small amounts of gas from glucose; the species name A. salmonicida is therefore retained. The genus Plesiomonas remains well defined with a single species, P. shigelloides, though many strains are 121

CHARACTERS OF GRAM-NEGATIVE BACTERIA

agglutinated by phase 1 Shigella sonnei antiserum and it may be more closely related genetically to the Enterobacteriaceae than to Vibrio and Aeromonas. Similarly, RNA sequencing suggests that Aeromonas could be separated from the Vibrionaceae into a new family, Aeromonadaceae (Colwell et aL, 1986). Under these proposals, the Vibrionaceae would contain Vibrio and Photobacterium as well as the new genera Listonella and Shewanella. The latter genus, Shewanella, would contain no pathogens but include some species previously placed in Alteromonas. However, unlike the other genera in this family, strains of Alteromonas are not active fermenters and we suspect their inclusion in such a new genus may not meet with general approval (but see Section 7.5). Taxonomists will no doubt continue to debate the generic allocation of species but for identification purposes and for convenience we still prefer to consider Vibrio, Aeromonas and Plesiomonas together because they share so many routine laboratory test characteristics; we also continue, for the same reasons, to consider Chromobacterium violaceum in this group since the production of a diffusible pigment may be no more than a species characteristic.

[7.8]

Vibrio species may also cause sporadic cases of diarrhoea as well as opportunist superficial eye, ear or wound infections. For the isolation of cholera and other vibrios from faeces, both direct plating and enrichment methods should be used. All the known enteropathogenic species, (except V. hollisae) grow on Thiosulphate Citrate Bile-salt Sucrose medium (TCBS) which is the selective agar medium of choice. After overnight incubation on TCBS Agar at 37 °C, large or mediumsized yellow (sucrose-fermenting) colonies should be subcultured onto Nutrient Agar, checked for purity, and tested for agglutination with V. cholerae Ol antiserum and for the oxidase reaction. This may be sufficient for recognition of an epidemic strain but otherwise biochemical characterization should be used for generic confirmation and species identification. With seafood and related environmental samples associated with food-poisoning, large yellow (sucrose-fermenting) colonies with a tendency to spread or swarm are more likely to be V. alginolyticus; a widespread halophilic and chitinolytic marine organism. Green (sucrose non-fermenting) colonies on TCBS medium should be regarded as probable strains of V. parahaemolyticus (Barrow & Miller, 1976). 7.8.1 Vibrio (Table 7.8a, b, c). At present, some 34 species of Vibrio have been validly described, Enrichment in Alkaline Peptone Water containing though knowledge of those recently approved is lim1% NaCl with early subculture from the surface to ited and many of them have not been isolated from TCBS or other selective agar media is suitable for all clinical material. They all require NaCl for growth, vibrios. For V. parahaemolyticus in food and envithe optimum concentration varying with the species; ronmental samples, however, selective enrichment in the requirement of some, such as V. cholerae, V. media such as Glucose Salt Teepol Broth or metschnikovii and V. mimicus, as well as some strains Polymyxin Salt Broth can be used (Barrow & Miller, of V. fluvialis, V. furnissii and V. anguillarum, is so 1976; Ayres & Barrow, 1978; Joseph, Kaper & low that they are able to grow, albeit suboptimally, in Colwell, 1982). Isolation of vibrios from clinical Tryptone Water without added NaCl. In contrast, material other than faeces is relatively easy as they all halophilic marine species such as V. alginolyticus and grow well on Blood Agar and other non-selective V. parahaemolyticus can tolerate concentrations of media. NaCl as high as 6-8%. All the medically important We show the characters useful for distinguishing species will, however, grow in isolation and identifiVibrio species from each other as well as from related cation media containing 1 % NaCl, as indeed do virtugenera in Tables 7.8a, b: the species listed have all ally all pathogenic bacteria. The differentiation of been encountered in clinical material or in associated arginine-positive Vibrio species from Aeromonas, food and environmental samples. For provisional however, depends on this salt requirement. The name identification vibrios are motile Gram-negative rods Vibrio usually implies gastrointestinal infection which ferment glucose without gas production, are caused by V. cholerae or by the marine organism V. sensitive to the vibriostatic agent O/129 (2-4parahaemolyticus, which is commonly associated diamino-6,7-diisopropylpteridine) and, except for V. with food-poisoning from sea-foods; but many other metschnikovii which is atypical, are oxidase-positive. 122

[7.8]

VIBRIO AND VIBRIO-LIKE GROUP

Table 7.8a. Second-stage table for Vibrio, Aeromonas, Plesiomonas, Chromobacterium and Janthinobacterium

Growth at 37 °C Motility Oxidase Nitrate reduced M0ller's decarboxylases arginine lysine ornithine Gas from glucose Resistance to O/129 10 jig O/129 150 jig Growth with 0% NaCl Growth with 6% NaCl

1

2

3

4

5

6

7

8

9

+ + + +

+ + + +

+ + -

+ + + +

+ d + +

_ + +

+ + + +

+ + + +

_ + + +

+ d -

+ d d

+ d -

-

+ d _ d

+ d w/-

+ + + -

+ -

-

d d d

d d d

d +

d d +

+ + + -

+ d + -

d d + -

-

-

?

?

-

9

-

1 Vibrio spp. including V. cholerae, V. mimicus, V. vulnificus, V. parahaemolyticus, V. alginolyticus, V. harveyi, V. cincinnatiensis 2 Vibrio spp. including V. fluvialis, V. furnissii, V. anguillarum, V. damsela 3 Vibrio spp. including V. metschnikovii 4 Vibrio spp. including V. hollisae, V. natriegens 5 Aeromonas spp. 6 Aeromonas salmonicida 7 Plesiomonas shigelloides 8 Chromobacterium violaceum 9 Janthinobacterium lividum Symbols used in the tables are explained in Tables 5.1 and 5.2 on p.47.

Although not taxonomically valid, Barrow & Miller (1976) found the catalase reaction and the methylene blue sensitivity test of Yan (1969) helpful for screening purposes. All vibrios have a DNA G+C mole% ratio of between 38 and 51 compared with 57-60 for Aeromonas (Veron, 1966; Hugh & Sakazaki, 1972) and most of them possess a single polar flagellum; these characters are of little use for differentiation in the routine laboratory where the key tests for this purpose include the O-F, VP and sugar reactions, Moeller's arginine, lysine and ornithine tests and those for nitrate reduction and salt tolerance. Taxonomically V. cholerae is a homogeneous species, comprising organisms that are similar to each other biochemically, share a common H antigen and are closely related genetically. Based on the somatic O antigen scheme of Sakazaki et al. (1970) more than 80 serogroups are now recognised within the species (Donovan, 1984), serogroup Ol representing the causal organism of epidemic and pandemic cholera. The cholera toxin (CT) responsible for the

'rice water' stools of cholera has been characterized, the gene concerned identified and a DNA probe developed for its recognition (Mekalanos, 1985). Although not yet applicable in routine laboratories, such a gene probe would enable the rapid screening of strains for toxigenicity. Some strains of other serogroups, termed 'non-Ol V. cholerae", can also produce cholera toxin. In contrast, in countries where cholera is not endemic, Ol strains, particularly from environmental sources, usually lack the CT gene. V. cholerae serogroups occur, sometimes in large numbers, in brackish and estuarine waters throughout the world; most of these environmental strains are incapable of producing CT, and they probably do not cause diarrhoea. Toxigenic strains may however also survive in such waters for many years (Miller, Feachem & Drasar, 1985; Bourke et al, 1986). Strains of 'non-Ol V. cholerae' may be isolated from sporadic cases of diarrhoea: indeed in countries where cholera is not endemic, such strains are more likely to be isolated than Ol strains from those 123

[7.8]

CHARACTERS OF GRAM-NEGATIVE BACTERIA

Table 7M. Third-stage table for Vibrio 1

2

3

4

5

6

7

8

9

10 11

12 13

14

Swarming Growth with 0% NaCl M0ller's decarboxylases: arginine lysine ornithine

+

_ +

_

d _

+ _

_

? _

_ d

_ d

_ d

7 -

_ +

7 -

_ _

+

_ + +

_ + d

_ + +

_ + -

+ -

+ -

+ -

+ d

+ d

-

+

_ + +

-

rNiirdies reuuccu

T

Oxidase Gas from glucose Indole ONPG VP Resistance to 0/129 10 |ag 0/129 150 jig ampicillin 10|ig polymyxin B 50 i.u. Hydrolysis of: starch urea Acid from: L-arabinose arbutin salicin sucrose xylose Growth on: ethanol propanol D-galacturonate D-glucosamine

+ + + +

+ + + -

+ _ + + -

+

+

+

+

+ +

+ d -

+ +

+ + — + -

+

+ -

d + -

+

-

d d

+ + d

+ d + -

d d + -

+ -

+ + -

+

-

+ —

+ d

+ d

+ d

+ -

+ -

d -

+ + d

d -

d d

d + d

-

-

-

d +

d d

-

+

+

+

d

-

+ +

+

1 Vibrio cholerae; V. comma'; cholera vibrio 2 Vibrio mimicus 3 Vibrio vulnificus 4 Vibrio parahaemolyticus; 'Beneckea parahaemolytica'; 'Oceonomonas parahaemolytica V 'Pastemella haemolytica '; 'Pseudomonas enteritis'

-

-

-

-

+

+

+ + +

— — + + + d -

+ d _

+ _ + _

d _ _ _ _ + ? d ? _ _ _ _

+ -

+ -

d _

+ ? + ? _ _ _ _

+ d

+ + + + +

+ d d + -

+ + _

d ? + _ _ _

+ d d + _

+ + + + -

? 7 ?

+ + + +

+ + + +

d

d d d

5 Vibrio alginolyticus; 'Oceonomonas alginolytica' 6 Vibrio harveyi 7 Vibrio cincinnatiensis 8 Vibrio fluvialis; V. fluvialis biotype I 9 Vibrio furnissii; V. fluvialis biotype II 10 Vibrio anguillarum

+ 11 12 13 14

? ? ? ?

Vibrio Vibrio Vibrio Vibrio

-

? ? ? ?

+

damsela metschnikovii hollisae natriegens

Data for V. cincinnatiensis from Brayton et al. (1986). Data for V.damsela and V. hollisae from Farmer, Hickman-Brenner & Kelly (1985). Remaining data from Bryant et al. (\986a,b) Symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

returning from warm climates. Although occasional non-Ol strains can produce cholera toxin most of them have a different enteropathogenic mechanism, as do non-CT-producing Ol strains which are occasionally isolated from patients with diarrhoea. Only Ol strains that produce cholera toxin seem to be associated with cholera epidemics whereas the majority of those isolated from the environment, whatever 124

their serogroup, appear to be incapable of causing diarrhoea. Thus, within the species V. cholerae as now defined, there is a continuous spectrum from enteropathogenic epidemic Ol strains at one extreme to non-pathogenic strains present in the environment at the other extreme. Taxonomists accordingly argue that the colloquial terms 'non-cholera vibrio' (NCV)

VIBRIO AND VIBRIO-LIKE GROUP

and 'non-agglutinable vibrio' (NAG) often applied in the past to non-Ol strains should no longer be used because of the variability in their toxigenicity, and because they are of course agglutinated by their homologous antisera. Also these terms have previously been used wrongly by the inclusion of other Vibrio species and even aeromonads within them. We certainly agree that the term 'non-agglutinable vibrio' (NAG) is inappropriate though we still think that 'non-cholera vibrio' (NCV) has as much if not more meaning medically (and microbiologically) than the awkward term 'non-Ol V. cholerae'. However, the clinical and epidemiological significance of vibrio isolates should in any case always be explained to clinicians. The epidemic cholera strains, V. cholerae Ol, can be divided into two variants commonly called the 'Classical' or cholerae biotype and the eltor (or El Tor) biotype. The eltor biotype was largely responsible for the pandemic in Asia which began in the 1960s and replaced the classical strains, until the latter reappeared. In general, eltor strains, are VPpositive: they haemolyse sheep erythrocytes in Thioglycollate Cysteine Agar (Sakazaki, Tamura & Murase, 1971) or in the tube test of Feeley & Pittman (1963); agglutinate chicken erythrocytes, in the slide test of Finkelstein & Mukerjee (1963); are sensitive to polymyxin B (50iu disc test of Gan & Tija, 1963); resistant to classical phage IV (Mukerjee, Guha & Guha-Roy, 1957); and sensitive to eltor phage V (Basu & Mukerjee, 1968). Classical strains have the opposite reaction in each of these tests. As they require careful standardization and adequate controls, these tests are more suitable for reference laboratories. The majority of non-Ol strains and Ol environmental isolates are strongly haemolytic and show the other characteristics of eltor strains except that they are usually resistant to both phages. Strains of V. cholerae Ol may be subdivided with absorbed sera into antigenic variants or subtypes called Ogawa, Inaba and Hikojima which share three somatic antigens (a, b and c) in different proportions. Absorption of 01 antiserum with Ogawa organisms produces a serum which agglutinates Inaba and Hikojima but not Ogawa strains. Similarly Ol antiserum absorbed with Inaba organisms agglutinates only Ogawa and Hikojima strains. Unabsorbed V. cholera Ol antiserum is still commonly but confus-

[7.8] ingly described as 'polyvalent V. cholerae antiserum' because it agglutinates all three of these Ol variants although it does not agglutinate strains of non-Ol serogroups. As there are only three antigenic variants, more than one of which may be isolated from the same patient (Sakazaki & Tamura, 1971), they have only limited, if any, value for epidemiological purposes. Nor are the fermentation groups of Heiberg (1936) of any use as they do not correlate with anything and they cannot distinguish between Vibrio and Aeromonas (Sakazaki, Gomez & Sebald, 1967). Infection with V. parahaemolyticus, a marine organism widely distributed in temperate and warm coastal waters throughout the world, is usually associated with inadequately cooked or raw seafoods; indeed, it is the commonest cause of food poisoning in Japan. First described by Fujino in 1951 (see Fujino, 1974) and regarded as a species of Pasteurella, it was later placed in the genus Pseudomonas and finally, following taxonomic studies, in Vibrio (Sakazaki, Iwanami & Fukumi, 1963; Fujino et al., 1965). Most strains isolated from patients with diarrhoea are Kanagawa-positive - that is, they are able to lyse human erythrocytes in Wagatsuma agar - in contrast to less than 1% of isolates from seafoods and environmental samples (Miyamoto et al, 1969; Peffers et al, 1973). Despite much research, the correlation between haemolysin production and pathogenicity is still unclear, although a laboratory infection and volunteer feeding tests convinced Sakazaki and his colleagues (1968) that Kanagawa-positive strains are potentially pathogenic. For epidemiological purposes, some strains may be further differentiated by their O and K antigens (Sakazaki & Balows, 1981) and commercial sets of antisera are available. Serologically, V. parahaemolyticus and V. alginolyticus have a common H antigen and there is some overlapping of O antigens (Sakazaki et al, 1968). V. mimicus (Davis et al., 1981) has the same H antigen as V. cholerae and strains of this species were originally regarded as sucrose-negative variants of it. However, DNA hybridization and phenotypic characterization confirm its separate identity. Unfortunately, true sucrose-negative strains of V. cholerae do occur occasionally but, unlike V. mimicus, they are VP-positive and resistant to Polymyxin B (50 iu). V. mimicus has been isolated occasionally from sporadic 125

CHARACTERS OF GRAM-NEGATIVE BACTERIA

cases of diarrhoea usually following the consumption of seafood; its enterotoxgenicity is similar to that of strains of non-01 V. cholerae. V.fluvialis (Lee et aL, 1981), previously described as 'group F' and as 'CDC group EF6' (Huq et aL, 1980) halophilic vibrios, comprises two biovars: I (anaerogenic) and II (aerogenic). However, based on DNA hybridization biovar II was assigned to a new species V. furnissii (Brenner et aL, 1983). The anaerogenic V. fluvialis occurs in fresh, brackish and estuarine waters throughout the world (Lee et aL, 1981). The circumstantial evidence for its enteropathogenicity is strong but, like V. parahaemolyticus, the mechanism has yet to be elucidated (Huq et aL, 1980; Lockwood, Kreger & Richardson, 1982). V. furnissii (Brenner et aL, 1983) is similar in habitat to V. fluvialis and has been isolated occasionally from patients with diarrhoea, though usually together with recognised pathogens; there is no direct evidence of its pathogenicity. Gas production from sugar differentiates it from V. fluvialis (Table 7.8c). The recently described halophilic species V. hollisae (Hickman et aL, 1982) isolated from faeces on Sheep Blood Agar may be missed because it does not grow on TCBS or MacConkey agar or on other commonly used media selective for intestinal pathogens; its pathogenic role is uncertain (Morris et aL, 1982). Other species, such as V. alginolyticus and V. metschnikovii, have also been detected occasionally in faeces but there is no evidence to implicate them as the causal agents of diarrhoea. Except for V. anguillarum, V. harveyi and V. natriegens, all the Vibrio species in Table l.Sb, including the halophilic species V. alginolyticus (Furniss, Lee & Donovan, 1978) and V. damsela (Love et aL, 1981; Morris et aL, 1982) have been isolated occasionally from opportunist tissue infections, which are usually superficial. V. vulnificus, previously known as 'Lactose-positive (L+) Vibrios' (Farmer, 1979) and briefly as 'Beneckea vulnifica (Reichelt, Baumann & Baumann, 1976), has been implicated occasionally in severe wound infections and septicaemia (Tison & Kelly, 1984). As it is similar to V. parahaemolyticus, reports prior to 1979 of such infections caused by the latter should be viewed with caution. Unlike V. parahaemolyticus, V. vulnificus ferments arbutin and salicin. V. cincinnatiensis 126

[7.8]

(Brayton et aL, 1986) was isolated from the blood and CSF of a patient with meningitis; similar organisms have also been isolated from animal faeces. Recent reviews on the identification and characterization of Vibrio species and related genera include those of Sakazaki & Balows (1981), Joseph, Kaper & Colwell, (1982), Lee & Donovan (1985) and Bryant et al. (1986a, b). Minidefinition: Vibrio. Gram-negative rods, motile. Aerobic and facultatively anaerobic. Catalase-positive. Oxidase-positive and reduce nitrate to nitrite (except V. metschnikovii). Attack sugars by fermentation; gas not produced. NaCl (Na+ ions) essential for growth. Type species: V. cholerae; NCTC strain 8021. 7.8.2 Aeromonas (Tables 7.8a, c). In contrast to Vibrio, the subdivision of Aeromonas into species still remains controversial, especially as the fish pathogen previously classified as 'Necromonas salmonicidd' is now retained within it. Excluding the latter, Cowan, in the previous edition of this Manual, agreed with Ewing, Hugh & Johnson, (1961) that there should only be a single species, A. hydrophila, although Eddy (1960, 1962) had distinguished two species, the aerogenic, VP-positive, gluconate positive 'A. liquefaciens \ and the anaerogenic VP-negative, gluconate-negative 'A. formicans'. Schubert (1961 a,b), however, regarded anaerogenic strains as varieties of three aerogenic species, which he labelled A. punctata, A. hydrophila, and A. salmonicida. Later, Schubert (1971) argued that the aerogenic species, 'A. liquefaciens', should be abandoned as it was not recognizable, and that the VP-positive A. hydrophila, which he described, should become the type species. The main difference between A. hydrophila and A. punctata (which Schubert had previously favoured as the type species) seemed to be only the production of acetoin (VP-positive) by the former. The strain described as a new species, 'A. proteolytica', by Merkel et al. (1964) is not an aeromonad but a Vibrio species correctly named V. proteolyticus. The current division of Aeromonas is based on motility and optimum growth temperature. The non-motile strains, which do not grow at 37 °C and are pathogenic for fish, fall into the genetically

[7.8]

VIBRIO AND VIBRIO-LIKE GROUP

Table 7.8c. Third-stage table for Aeromonas, Plesiomonas and Vibrio (V. fluvialis and V. furnissii)

Motility Growth in 0% NaCl Growth in 6% NaCl M0ller's decarboxylases: arginine lysine ornithine Gluconate oxidation Gas from glucose Indole VP Resistance to O/129 10 Jig O/129 150|ag Hydrolysis of: aesculin lecithin Acid from: L-arabinose arbutin inositol salicin sucrose Growth on: ethanol propanol D-galacturonate D-glucosamine

1

2

3

4

5

6

7

8

+ + -

+ + -

d + -

d + -

d + _

+ d +

+ d +

+ + -

+ + + d + +

+ + d d + d

+ d -

+ + d d -

+ d d d

+ _ d -

+ + _ -

+ + + + -

+ d

+ +

+ +

+ d

+ d

+ -

+ -

+ -

+ d

+

d d

d +

_

d +

+

-

d + d + +

d +

+ d d +

9

+ d d

d d +

+ d d +

+ +

d _

+

+

d +

-

-

+ + + +

+ + + +

+

1 Aeromonas hydrophila; 'A. liquefaciens', 'A. punctata'; 'A. formicans' 2 Aeromonas sobria 3 Aeromonas caviae 4 Aeromonas salmonieida subspecies salmonicida; 'Necromonas salmonicida' 5 Aeromonas salmonicida subspecies masoucida and achromogenes; 'Necromonas salmonicida' 6 Vibrio fluvialis; Vibrio fluvialis biovar I 7 Vibrio furnissii; Vibrio fluvialis biovar II 8 Plesiomonas shigelloides; C27; 'Aeromonas shigelloides'; 'Vibrio shigelloides'; 'Fergusonia shigelloides'; 'Pseudomonas michigani' Symbols used in this table are explained in Tables 5.1 and 5.2 on p.47.

homogeneous species, A. salmonicida, which has three phenotypic subspecies: salmonicida, masoucida and achromogenes. A proposed new species, A. media, found in the environment is also non-motile and is described as intermediate between A. hydrophila and A. salmonicida (Allen, Austin & Col well, 1983). As neither A. salmonicida nor A. media causes disease in man we disregard them,

although for comparison with other species we include the former in Table l.Sb. The majority of motile strains, which grow well at 37 °C, fall into three phenotypic groups corresponding to those described by Popoff & Veron (1976) called A. hydrophila, A. sobria and A. caviae: they can be differentiated by the production of acid from salicin and gas from glucose. DNA hybridization has demonstrated other groups but these cannot be otherwise distinguished (Popoff et al., 1981). However, in the Approved Lists of Bacterial Names (1980) A. punctata is listed with two subspecies: caviae and punctata', and the DNA studies of Popoff et al., (1981) also revealed two hybridization groups in the 'caviae-punctata' group. We therefore leave it to taxonomists to resolve the nomenclatural complexities in this area and refer only to A. caviae in Table 7.8c. The motile aeromonads are common in freshwater environments where they are frequently the dominant aerobic bacteria. They have been isolated from a wide range of clinical infections of wounds and soft tissues, sometimes following exposure to water, and from some patients with diarrhoea in the absence of recognised pathogens as well as from septicaemia in immunocompromised patients. Their possible enteropathogenic role has received much recent attention (Pitarangsi et al., 1982; Burke et al., 1983) and a number of selective media have been described for their isolation from faeces (von Graevenitz & Bucher, 1983; Lee & Donovan, 1985). Aeromonas species, in particular A. caviae, may be confused with strains of Vibrio fluvialis and V. furnissii but resistance to the vibriostatic agent O/129 (150|Lig disc) and growth in the absence of NaCl as well as growth of the vibrios in the presence of 6% NaCl should resolve the matter. Minidefinition: Aeromonas. Gram-negative rods. Motile and non-motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-positive. NaCl (Na+ ions) not required for growth. Sugars are attacked fermentatively and gas may be produced. Type species: A. hydrophila; NCTC strain 8049. 7.8.3 Plesiomonas (Tables 7.8a,c). The existence of this genus was due almost entirely to uncertainty about the allocation of its single species, P. 127

CHARACTERS OF GRAM-NEGATIVE BACTERIA

shigelloides, which has been placed previously in Aeromonas, Vibrio and Pseudomonas. Originally referred to as the C27 organism of Ferguson & Henderson (1947), it was moved to the now valid genus Plesiomonas created for it by Habs & Schubert (1962) and supported by Eddy and Carpenter (1964). Its specific name is based on the observation that many strains are agglutinated by phase 1 antiserum to Shigella sonnei, from which they can be distinguished by their motility and positive oxidase reaction. They can be differentiated readily from aeromonads and vibrios by O/129 resistance, fermentation of inositol and the production of ornithine decarboxylase and DNase. Most P. shigelloides strains are isolated from patients with diarrhoea, often in the absence of known pathogens (Pitarangsi et al, 1982); other infections in children have been summarized by Pathak, Caster & Levy (1983). Minidefinition: Plesiomonas. Gram-negative rods; motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-positive. Sugars attacked by fermentation; gas is not produced. Inositol is fermented and gelatin not liquefied. Ornithine decarboxylase-positive (M0ller). Type species: P. shigelloides; NCTC strain 10360. 7.8.4 Chromobacterium (Table 7.8a) is a genus with a single mesophilic species, C. violaceum, that may be isolated occasionally in the clinical laboratory. Like the psychrophilic organism, Janthinobacterium lividum, it produces a violet pigment, violacein, that is soluble in ethanol, acetone and amyl alcohol, but is insoluble in chloroform and in water and therefore does not diffuse through the medium. Production of violacein is encouraged by the presence of mannitol and yeast extract in the medium but the pigment makes the oxidase test difficult to read; it is produced only in the presence of abundant oxygen (Sneath 1960). The older descriptions of C. violaceum are confusing because the name was given to nearly all cultures that produced a violet pigment. Sneath (1956) applied the name correctly to the mesophiles; but Leifson (1956) and Eltinge (1956, 1957) used it for the psychrophile now called Janthinobacterium lividum. Apart from differing in the temperature range for growth, J. lividum has an oxidative metabolism and 128

[7.8]

we think it is therefore correctly placed with the other oxidizers in Table 7.5; for comparison with the mesophilic organisms, however, we include its characters also in Table 1.8a. C. violaceum may occasionally be pathogenic for man (Sneath, 1960; Johnson, Disalvo & Stener, 1971) causing abscesses, urinary tract and systemic infections and possibly diarrhoea. The likely source is from soil or water but this has never been clearly demonstrated. Minidefinition: Chromobacterium. Gramnegative rods, motile. Aerobic and facultatively anaerobic. Catalase-positive (usually); oxidase-positive. Attack sugars fermentatively. Type species: C. violaceum; NCTC strain 9757. 7.9

The enterobacteria (Buttiauxella, Cedecea, Citrobacter, Edwardsiella, Enterobacter, Erwinia, Escherichia, Hafnia, Klebsiella, Kluyvera, Morganella, Proteus, Providencia, Salmonella, Serratia, Shigella, Tatumella, Yersinia and others)

Characters common to members of the group: Gram-negative rods. Aerobic and facultatively anaerobic. Oxidase-negative. Attack sugars fermentatively. Nitrate reduced to nitrite. The enterobacteria grow readily on simple media, on which they will survive for years in tubes sealed with paraffin wax. The majority of species, being easy and 'safe' to handle, provide students, biochemists, geneticists, and even bacteriologists with suitable experimental material. Nevertheless, laboratory infections do occur, especially with Shigella, which has a very low infective dose. Because some strains are important pathogens for man and animals and cause intestinal and other infections as well as food-poisoning, the group has considerable epidemiological importance and means are available to 'fingerprint' the members precisely. They have been studied exhaustively by numerous bacteriologists and have introduced many to taxonomy; they have provided feasts for the splitters and banquets for the geneticists. As a result they have been divided into a large number of genera and species, some of which have

ENTEROBACTERIA

been further subdivided into countless serotypes and phage types, but, as Kampelmacher (1959) pointed out, there are no antigenic frontiers among the enterobacteria and there is a great deal of overlapping. The whole group corresponds to what was 'Bacterium' before that genus became the dumping ground for bacteria that could not be assigned readily to some other genus. Nowadays it it usual, if not meaningful, to split the enterobacteria first into tribes and then into genera. In a review of the group, Cowan (in Bergey's Manual, 191 A) speculated that what are now regarded as tribes may become the genera of the future as he considered that there had been an excessive subdivision of the group into genera and species; however, recent DNA data now largely confirm the validity of these subdivisions. In this Manual we do not make the division into tribes; we have taken genera and species that will be accepted by most (but not by all) bacteriologists and show some characters (not important in themselves) by which they can be distinguished. A second-stage table (7.9a) leads on to four third-stage tables {1.9b, c, d, e) by which an unknown strain of the group can be identified down to species level in the group. The classification of the enterobacteria is by no means final and the divisions we make do not always correspond to those made by others. Numerous synonyms are given to assist the reader in relating current names to older literature. To emphasize that this arrangement is devoid of phylogenetic or hierarchical implications, the notes for the genera have been arranged in alphabetical order. Although the tables are useful in aiding identification down to species level as it is understood in this group, this level is well below that of species in many other large groups of bacteria such as the corynebacteria and streptococci. This picture may well change, however, as other large bacterial groups are studied as intensively as the enterobacteria. We do not proceed to biotypes or to the finer subdivisions made by serological and phage-typing methods since few clinical laboratories will have a sufficiently wide range of antisera or phages for such work. Fortunately, reference laboratories are available in many countries, and even if they are national, the services are usually international. An entirely different means of arriving at an identification of enterobacteria in stages was described by

[7.9] Baer & Washington (1972). In this method individual characters are scored (e.g. acid from mannitol = 5; gas from mannitol = 10; acid from lactose = 20; growth in KCN medium = 320), and the total score is looked up in one or more identification tables. Such numerical diagnostic keys are discussed in Chapter 8; they have also been extensively reviewed by D'Amato, Holmes & Bottone, (1981). During the course of intestinal infections, the enterobacteria may be isolated from the bloodstream, intestinal contents (bile or faeces), often from urine, and occasionally from marrow biopsies. In the intestine they form part of a mixed bacterial flora, and are usually isolated by means of enrichment and selective media, from which pure cultures for identification must be obtained by plating on non-inhibitory media. 7.9.1 'Aerobacter' is a name that has caused much confusion because it has been applied to several different organisms. Originally used by Beijerinck (1900) for organisms (motile or nonmotile) that had optimal growth temperatures about 28 °C but which would not grow at 37 °C, it was later used extensively for organisms that grew at 37 °C or higher, and were usually non-motile. In 1958 Hormaeche & Edwards, who wanted to distinguish klebsiellas from motile bacteria with similar IMViC reactions, used the name for a motile organism that grew at 37 °C; when they realized the difficulties arising from the use of the same name for two different organisms, they changed the name of the group of motile bacteria to Enterobacter (Hormaeche & Edwards, 1960). Carpenter and many others (1970) asked the Judicial Commission to give an opinion on the use of the name 'Aerobacter' and in Opinion 46 (1971) the Commission declared the name rejected. 7.9.2 'Bacterium', as a generic name, has been outlawed for many years (Opinion 4, revised, 1954) and it is now no longer used. The reason for its rejection was the different usage in different countries, although in any one country its use was probably well known and understood. In British practice it was traditionally used for Gram-negative rods that essentially comprised the enterobacteria; in the sixth edition of Bergey's Manual (1948), which was typical of American practice at that time, the genus 'Bacterium' 129

[7.9]

CHARACTERS OF GRAM-NEGATIVE BACTERIA

Table 7.9a. Second-stage table for differentiation of the enterobacteria

Motility Yellow pigment Red pigment MacConkey growth Simmons' citrate Christensen's citrate Urease Gelatin hydrolysis Growth in KCN medium H2S (PbAc paper) H2S from TSI Gluconate Malonate ONPG Phenylalanine Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Selenite reduction Casein hydrolysis DNase production Carbohydrates [in Peptone Water medium], gas from glucose acid from: adonitol arabinose cellobiose dulcitol glycerol inositol lactose maltose mannitol raffinose rhamnose salicin sorbitol sucrose trehalose xylose starch MR test (37 °C)« MR test (RT)* VP test (37 °C)« VP test (RT)* Indole

2

3

4

5

6

7

8

9

+ -

+ -

+ -

+

+ D

+ d

+ D

d

- 4 - 4 -

4-

4"

d + + _ + +

D + D _ D D +

+ -

+ + d D d D D +

+ -

+ D D

-

-

-

-

D

4-

_ + + d +

_ _ + D D

+ + -

+ + D D D _ + d +

d + _ d d _ d +

D D _ D D _

4-

4-

D D

+

4-

4-

d

+

D D D +

-

d

-

D

-

+

4-

D

-

4-

d

d + _

D + + d D + + + + + + D + + + D d d -

+ + 4-

44D

+

-

+

4-

-

D D

4-

4-

+ D d + + + D + D + d +

4-

D -

4-

-

4-

4-

+ -

D D D -

4+ d

+ + +

Further details in Table 1 2 3 4 5 6 7 8 9

10 11 12 13 14 15C \6d 17 1 *

1

Buttiauxella agrestis Cedecea spp. Citrobacter spp.; Levinea spp. Edwardsiella tarda; Asakusa biotype; Bartholomew group Enterobacter spp. Erwinia herbicola; Enterobacter agglomerans Escherichia spp. Hafniaalvei Klebsiella spp.

7.9b 10 11 12 13 14 15 16 17 18

+ +

d

D

-

4-

D d _ D D D

+ d _ + +

+ + _ -

4-

D D

d

-

4-

- 4 - 4 -

+

d

4-

d

-

-

_

_

-

4-

4-

D

+

_ + d d _ d d + d + + d + +

D + D D D _ d + + D + d D D +

_

4-

-

+ + D

+ + -

4-

d

d

4-

-

-

D +

+ +

-

44444d

4444d 4-

-

+

+

-

4-

4-

4-

4-

-

d d d -

+ D

+ d + _ _ + + _ + d d + + _ d d + -

d d d

— — 4- 44- 4-

D D D

d

D +

4-

d + _ + d _ + d +

D +

d

7.9c

D d + + + 4-

d

D

4-

4-

d + D + d

d -

D 4-

D D d d D D

4-

_

D

-

-

444-

4-

4-

D

-

4-

4-

-

-

_

_

-

-

d D _ + D +

_

-

-

d

-

d d d -

-

-

d D D

d d

4-

_

D d D +

D

D D d d D

+

4-

D

D

D

-

-

-

D

-

-

_ D D D D D D D + D D D D D -

D D d -

4-

4-

-

D D d + -

d D D _ D D d D _

4-

4-

d

+ +

-

+ _ D -

D D

44-

7.9d

44-

D 44-

D D

-

D D D D _ + D D

_ -

-

d d

-

4-

-

d D

-

4-

D 4-

D D 44-

D

-

-

4-

D D D D D

4-

4-

4-

-

4-

4-

-

4-

4-

+

D

-

+ D D

4-

-

7.9,

Kluyvera spp. Morganella morganii; Proteus morganii Proteus spp. Providencia spp. Salmonella spp. and serotypes Serratia spp. c Shigella spp.^ Tatumella ptyseos Yersinia spp. c

RT, room temperature (18-22 °C);a incubation for two days; b incubation for five days. c Some strains nitrate-negative. ^Some strains catalase-negative. Other symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

u

-

4-

D D + D d D D -

D

ENTEROBACTERIA

included both Gram-positive and Gram-negative rods. Most organisms that were formerly called 'Bacterium this-or-that ' in medical laboratories will be found in this Manual in Section 7.9 among the enterobacteria. Of the few exceptions, 'Bacterium anitratum* will be found in Section 7.4.2 under the awkward name of Acinetobacter calcoaceticus\ and 'Bacterium typhiflavum* is now placed in Erwinia (Section 7.9.8) as Erwinia herbicola (Graham & Hodgkiss, 1967). 7.9.3 Buttiauxella (Tables 7.9a, b). There is but a single species, B. agrestis, strains of which have been isolated mostly from water and soil but also from the throat and from urine. They resemble those of Citrobacter freundii in their IMViC reactions but differ from them in utilizing malonate, hydrolysing aesculin and usually in failing to ferment sorbitol (Ferragut et al, 1981). Strains of B. agrestis also closely resemble those of Kluyvera (see Section 7.9.12 and Tables 1.9a, c) but the DNA characteristics show that they should be maintained as separate genera, which Gavini et al. (1983) claim can be differentiated on the basis of indole and lysine decarboxylase production and fermentation of sucrose. Minidefinition: Buttiauxella. Gram-negative rods, motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Attack sugars fermentatively; gas is not produced. Sucrose-negative. KCN-positive; arginine-negative. Type species: B. agrestis; NCTC strain 12119. 1.9 A Cedecea (Tables 1.9a,b). Originally two named species, C. davisae and C. lapagei, were described together with three unnamed species referred to as Cedecea species 3, 4 and 5 (Grimont et al., 1981). The name C. neteri was later proposed for species 4 leaving species 3 and 5 unnamed (Farmer et al., 1982). All strains have been isolated from clinical material, mostly from patients with bacteraemia (Farmer et al, 1982) and from a cutaneous ulcer (Hansen & Glupczynski, 1984). In their biochemical reactions strains of Cedecea do not resemble any other member of the Enterobacteriaceae. They do not ferment arabinose or rhamnose or liquefy gelatin nor do they produce extracellular deoxyribonuclease;

[7.9] they are also resistant to colistin and cephalothin. Minidefinition: Cedecea. Gram-negative rods; motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Attack sugars fermentatively. Arabinose-negative. Arginine-positive. Type species: C. davisae; NCTC strain 11645. 7.9.5 Citrobacter (Tables 1.9a,b). The common water and soil forms, mainly Citrobacter freundii, are rapid lactose fermenters; less common varieties produce indole and may belong to C. freundii or more likely to C. amalonaticus (originally described as Levinea amalonatica by Young et al., 1971) or to C. koseri (see below). The Ballerup and Bethesda groups were non- or late-lactose fermenters of the citrobacter group, and in plate cultures could be recognized by the characteristically foul odour they produced. Sedlak and his colleagues (1971) in a review of 8000 strains of the genus regarded them as 'conditional' or opportunist intestinal pathogens when conditions were suitable for their growth in an unusual situation. The genus has an unusual history. It was created by Werkman & Gillen (1932) for bacteria that produced trimethylene glycol from glycerol; as tests for this substance were complicated, no-one ever used them but paid attention instead to the character (utilization of citrate as a source of carbon) after which the genus was named. Sedlak et al. (1971) recognized two species: Citrobacter freundii, H2S positive, indole-negative, and malonate-positive, and ' C intermedius" with the opposite characters; there were also many intermediate forms, which account for the seven species listed by Werkman & Gillen. Of those seven species, only Citrobacter freundii became established. Ewing & Davis (1912a) would revive C. diversus for the organism named C. koseri by Frederiksen (1970) and Levinea malonatica by Young et al. (1971). We have never been happy with this proposal, however, as the description of C. diversus given by Ewing & Davis (1972a) differs in several respects from the original description (Holmes et al, 191 A). We prefer to use the name C. koseri. Moreover, 'C. intermedius' is no longer recognized as a separate species. Chemical analysis of the lipopolysaccharides of 131

CHARACTERS OF GRAM-NEGATIVE BACTERIA

the O antigens indicates that citrobacters are 'closely related to E. colV and that, of twenty citrobacter chemotypes identified, eleven were 'identical with chemotypes occurring in Salmonella and E. colV (Keleti et ah, 1971). Perhaps citrobacters should be considered as a connecting link uniting Escherichia and Salmonella; if that is a fair description of the situation then bacteriologists can be excused for wanting a simple test for differentiating Citrobacter from Salmonella. Catsaras & Buttiaux (1963) thought that the lysine decarboxylase and KCN tests were useful for this purpose; citrobacters were lysine-negative and generally KCN-positive; salmonellas were lysine-positive (with a few exceptions) and KCNnegative. Pickett & Goodman (1966) suggested the ONPG test, but this also had its exceptions. We still think, however, that lactose fermentation is useful (Table 7.9a). Minidefinition: Citrobacter. Gram-negative rods; motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Attack sugars fermentatively; gas is produced. Citrate-positive; lysine-negative. Type species: C freundii; NCTC strain 9750. 7.9.6 Edwardsiella (Tables 7.9a,b). The taxon was first described by Sakazaki & Murata in 1962 (in Japanese) and by Sakazaki (1967), in English, as the 'Asakusa group'. It was redefined by King & Adler (1964) as the 'Bartholomew group', and elevated to a genus by Ewing et al. (1965) who also proposed the type species E. tarda. A new combination, Edwardsiella anguillimortifera, has been proposed for an organism formerly known as 'Paracolobactrum anguillimortiferum' and considered to be an earlier synonym of E. tarda (Sakazaki & Tamura, 1975); they have the same type strain. We continue to use the name E. tarda because it has been in common usage for so long. The features that distinguish Edwardsiella from Escherichia are mainly quantitative; it produces H2S in TSI whereas Escherichia coli is negative (but may be H2S-positive if tested by a more sensitive method). Ewardsiella tarda ferments fewer sugars more slowly (hence the species name), and a greater proportion of strains decarboxylate lysine and ornithine. In terms of DNA relatedness, however, Edwardsiella tarda 132

[7.9]

forms a distinct homogeneous group (Brenner, 1973). Other biotypes of E. tarda and other Edwardsiella species have not been isolated from clinical material; for their descriptions see, for example, Holmes & Gross (1983, 1990). Minidefinition: Edwardsiella. Gram-negative rods; motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Attack glucose fermentatively; produce gas. H2S produced on TSI medium. Type species: E. tarda; NCTC strain 10396. 7.9.7 Enterobacter (Tables 7.9a, b) was the name proposed by Hormaeche & Edwards (1960) for a group of motile organisms with IMViC reactions h +, and Enterobacter cloacae {'Bacterium cloacae' Jordan, 1890; 'Cloaca cloacae' Castellani & Chalmers, 1919) was designated as the type species. Other organisms including Enterobacter aerogenes, E. gergoviae, E. sakazakii and E. taylorae have since been placed in the genus. Despite its motility, numerical taxonomic studies of biochemical characteristics (Bascomb et al., 1971) and DNA relatedness (Izard et al., 1980) both indicate that E. aerogenes is related more closely to Klebsiella species than to the other Enterobacter species. The name Klebsiella mobilis has therefore been proposed for E. aerogenes but it has not been widely accepted. E. gergoviae strains were recognized by Richard et al. (1976) as a distinct group most similar to E. aerogenes though differing from it in producing urease, not growing in KCN medium and in not fermenting adonitol, inositol and sorbitol; the species name gergoviae was proposed for them later by Brenner et al. (1980b). The name E. sakazakii was given to yellow-pigmented strains which failed to ferment sorbitol; such strains were for long regarded as variants of E. cloacae until genetic studies showed that they should be recognized as a separate species (Farmer et al, 1980). Although E. taylorae occurs occasionally in clinical material, we have not examined representative strains and so do not record its characteristics in the Tables. They are described (Farmer et al., 1985a) as giving positive results in the following tests: motility, citrate, KCN, malonate, arginine, ornithine, Voges-Proskaiier and fermentation of glucose (with gas production), arabinose, cellobiose, maltose, mannitol, rhamnose, trehalose and xylose; and negative results for indole,

[7.9]

ENTEROBACTERIA

Table 1.9b. Third-stage table for the enterobacteria (part 1)

Motility Yellow pigment Red pigment MacConkey growth Simmons' citrate Christensen's citrate Urease Gelatin hydrolysis Growth in KCN medium H 2 S (PbAc paper) H 2 SfromTSI Gluconate Malonate ONPG Phenylalanine Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Selenite reduction Casein hydrolysis DNase production Carbohydrates [in Peptone Water medium], gas from glucose acid from: adonitol arabinose cellobiose dulcitol glycerol inositol lactose maltose mannitol raffinose rhamnose salicin sorbitol sucrose trehalose xylose starch MR test (37 °C)fl MR test (RT)*7 VP test (37 °C)a VPtest(RT)^ Indole 1 2 3 4 5 6 7 8

1

2

+ _

+ _ _ +

+ d + _ + _ _ — + +

-

+ _ + _ _ — + + _ + _ _ + + - d _ _ _

3

4

5

6

+ + + + _ _ _ _ _ _ _ _ _ _ + + + + + + + + + + + d _ _ _ _ _ + + - _ _ _ _ d _ _ _ _ _ + d + — — + + + + + + + _ _ _ _ _ + + + + _ _ _ _ _ - - + + + + _ _ _ _ _ _ _ _ _ _ -

11

12

13

14

+

+

+

_ _

+

+ _ _ + + + d _ + -

d _ _ + + +

+

+ + _ + + +

+

+

+

+

+ + +

+ + _ _ _ d + d _ _ _ d d + + + + + + + d + + + + + + + + + + + + + + + + + + _ _ _ + d d d d d -

_ _

_ + +

_ _ _ _ _ _ _ + _ + + + d + + + + + + + + _ _ _ _ _ _ _ _ ( j _ d d d + - d + + d _ _ _ _ _ _ _ _ _ d d + + + + + + + + + + + + + + + + + + + + + d + d + + + + + + + + + + d + + + + + + + + + d d + + + + + + + + + + + + + + + _ _ _ _ _ _ _ _ _ _ + d + + + + + + + + + + + + _ + _ _ _ _ _ _ _ _ + d _ _ _ _ _ + d + +

Buttiauxella agrestis Cedecea davisae Cedecea lapagei Cedecea species 3 Cedecea neteri; Cedecea species 4 Cedecea species 5 Citrobacter amalonaticus; 'Levinea amalonatica' Citrobacter freundii 'EscherichiafreundiV; 'Salmonella coW; 'S. ballerup'; 'S. hormaechei'; Bethesda-Ballerup group

+

_ _

+

10

15

+ d _ _ + + + + + + + + + + d + + + + + + d d + - _ _ _ _ _ + _ + + - + + d + + + d + _ + _ _ _ _ _ — — + + + + + + + + d d + + + + + + + + _ _ _ _ _ _ _ _ _ + d + - + + _ _ + + _ d _ _ + d d + + + + + + + + d + + + + _ _ _ _ _ _ _ _ _ _ _ _ _ _ _ + _ _ _

+

9

-

+ + _ _ _

d

+

8

-

d _ + + + + + + + +

+

7

+

d

+ +

_ + + + + + + + + + + + + _ d

d d

d -

-

+ +

+ d _ d d d + d + + d + + + _ d d d -

9 Citrobacter koseri; C. diversus; Levinea malonatica 10 Edwardsiella tarda; E. anguillimortifera;Asakusa group; Bartholomew group 11 Enterobacter aerogenes; Klebsiella mobilis; (NOT 'Aerobacter aerogenes' Beijerinck) 12 Enterobacter cloacae; 'Cloaca cloacae'; 'Aerobacter cloacae' 13 Enterobacter gergoviae 14 Enterobacter sakazakii 15 Erwinia herbicola; 'Bacterium typhiflavum', Enterobacter agglomerans; Pantoea agglomerans;

RT, room temperature (18-22 °C);a incubation for two days; b incubation for five days. Other symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

CHARACTERS OF GRAM-NEGATIVE BACTERIA

urea, gelatin, H2S (TSI agar), phenylalanine, lysine, methyl red and fermentation of adonitol, inositol, raffinose and sorbitol. In American literature, the name Enterobacter agglomerans is generally used for the organism we describe below (Section 7.9.8) as Erwinia herbicola. The characters of Enterobacter species are very similar to those of Hafnia and Serratia, and Edwards & Ewing (1972) include Hafnia alvei (7.9.10) in Enterobacter. The two best-known species, E. cloacae and E. aerogenes, are only weakly urease-positive, thus enabling Matsen's (1970) ten-minute paperstrip test to distinguish between Enterobacter (negative) and Klebsiella (positive), since it depends on the relative inactivity of the Enterobacter species. Minidefinition: Enterobacter. Gram-negative rods; motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Attack sugars fermentatively; gas is produced. VP-positive; glueonate-positive. Gelatin may be liquefied slowly. Produce ornithine decarboxylase. Type species: E. cloacae; NCTC strain 10005. 7.9.8 Erwinia (Tables 1.9ajb). This genus was named by Winslow et al. (1917) after Erwin F. Smith, a plant pathologist who did not agree with and continually argued against the idea of using plant pathogenicity as the keystone for creation of a new genus. We think that certain bacteria isolated from human and other animal sources are so similar to organisms isolated from plants that they should all be included in the same genus; as the plant organisms are part of a recognized genus, Erwinia, and since the strains from mammals were never properly classified (until the proposal of the name Enterobacter agglomerans by Ewing & Fife, 1972; see below) they should all be regarded as Erwinia species. Moreover, Lakso & Starr (1970) found that about a third of the animal strains they tested were as pathogenic for plants as some of the species considered to be natural phytopathogens. Strains previously labelled 'Bacterium typhiflavum* (Cruickshank, 1935), which for many years could not be placed satisfactorily among the usual 'medical' bacteria, are now regarded as Erwinia herbicola (Graham & Hodgkiss, 1967). Similar organisms have been isolated in many clinical laboratories 134

[7.9]

but the characterizations do not always agree with those of the plant pathologists. Such discrepancies may be due to differences in media and temperature of incubation; for example, Ewing & Fife (1972) found that about a quarter of their strains produced gas from glucose, and their characterizations made them allocate their strains instead to Enterobacter as Enterobacter agglomerans. However, other workers report the human and animals strains as anaerogenic and these results point less clearly to Enterobacter. In any case, we are reluctant to accept the proposal of Enterobacter agglomerans because the description of the species differs from the original description of 'Bacillus agglomerans' in several important features; in particular, the latter was described as having polar flagella. Although Ewing & Fife (1972) recognized seven anaerogenic and four aerogenic biotypes within E. agglomerans this classification showed little correlation with DNA data, by which 10 or more groups could be identified within the species (Brenner et al., 1984). Additional studies are therefore needed before definite proposals for the classification and nomenclature of these organisms can be accepted. Until then we prefer to regard this heterogeneous group as a single species with the name Erwinia herbicola. Although we are not here concerned with the classification and identification of the phytopathogenic erwinias, it should be noted that not all is neat and tidy; indeed Starr & Mandel (1969) are agnostic about the genus. Fermentation end-products merely support the argument that Erwinia is a member of the Enterobacteriaceae (White & Starr, 1971), but for the phytopathogens they do not provide any better guide to species than they do for the animal strains. One unusual character not shown in the tables is the symplasmata (sausage-shaped aggregates of bacteria) seen in hanging-drop preparations of the water of condensation from slope cultures (Gilardi, Bottone & Birnbaum, 1970a). Minidefinition: Erwinia. Gram-negative rods; motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Attack sugars fermentatively\ usually without producing gas. Urease-negative; phenylalanine-negative; KCN-negative. Type species: E. amylovora; ATCC strain 15580.

ENTEROBACTERIA

7.9.9 Escherichia (Tables 7.9a,c). Escherichia is the 'type genus' for the family Enterobacteriaceae and Escherichia coli is the type species for the genus. E. coli is possibly the most studied as well as one of the more important bacterial pathogens. In addition, the genus includes the less important species E. adecarboxylata, E. blattae, E. fergusonii, E. hermanii and E. vulneris. Escherichia coli, like many other enterobacteria, contains numerous serotypes some of which are associated with certain infections in man and in animals. Some are particularly associated with diarrhoeal disease (Gross & Rowe, 1985; Morris & Sojka, 1985) while others cause a variety of extra-intestinal infections (0rskov & 0rskov, 1985). The species includes a wide range of biochemical varieties, from the 'typical' motile, aerogenic, lactose-fermenting strains to the much less reactive and frequently non-motile strains that closely resemble Shigella. Such strains may be difficult to distinguish from shigellas and tests for the utilization of citrate (Christensen's medium) and the decarboxylation of lysine are particularly useful for this purpose. These problematic strains also include the Alkalescens-Dispar (A-D) group, a term previously used for organisms named 'B. alkalescens" and 'B. dispar' by Andrewes (1918) and which were once thought to be shigellas. They are now regarded as anaerogenic (non-motile) biotypes of E. coli (Ewing, 19496); the old term A-D is no longer used. It is not surprising that the distinction between E. coli and Shigella may be difficult as Brenner et al. (1982c) have shown by DNA hybridization that the two organisms belong to a single genetic species. To make the distinction even more difficult, there is considerable antigenic overlap between E. coli and Shigella so that virtually every Shigella serotype has a corresponding E. coli O group that is either related or identical to it (Edwards & Ewing, 1972; Rowe, Gross & Guiney, 1976). An operational classification based on indole and gas production from lactose at 44 °C together with other IMViC reactions, despite its limitations, is still used for water examination purposes (Report, 1956c, 1969, 1983). On first isolation many strains of Escherichia are non-motile or only feebly motile and for this reason motility is shown as a 'd' character in Table 7.9c.

[7.9] Escherichia adecarboxylata was described by Leclerc (1962). Strains are indole-positive, lysine decarboxylase-negative and usually produce a yellow pigment. The species has received little attention but strains are occasionally isolated from clinical specimens. Ewing & Fife (1972) included E. adecarboxylata in Enterobacter agglomerans (= Erwinia herbicola) (see Section 7.9.8); the species is certainly misplaced in Escherichia and a new genus Leclercia has been proposed to accommodate it (Tamura et al., 1986). Escherichia blattae, isolated from the intestinal tract of cockroaches, has not been reported in clinical material and therefore we do not include it in Table 7.9c. The species, which is indole-negative and nonmotile, was described by Burgess, McDermott & Whiting, (1973); we suspect that it is probably misplaced in Escherichia. Escherichia fergusonii, isolated from animals and human clinical material, was proposed by Farmer et al. (19856) to include a group of motile, non-lactosefermenting strains which are indole- and lysine decarboxylase-positive. They differ from E. coli in their ability to ferment cellobiose. Escherichia hermanii, isolated particularly from wounds, was described by Brenner et al. (1982a) to include motile, indole-positive strains which ferment cellobiose, grow in KCN and .may produce a yellow pigment on suitable media; lactose fermentation and lysine decarboxylase production vary from strain to strain. Escherichia vulneris was also described by Brenner et al. (19826) as a new species to include a group of strains many of which were isolated from human wounds. They are motile, indole negative, lysine decarboxylase positive and ferment cellobiose; lactose fermentation is either delayed or absent. Mini definition: Escherichia. Gram-negative rods; often motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidasenegative. Attack sugars fermentatively; gas normally produced. Usually citrate-negative (Simmons' medium) (except some E. blattae strains). Type species: E. coli; NCTC strain 9001. 7.9.10 Hafnia (Tables 7.9a, c). The Hafnia group was described by Stuart & Rustigian (1943a ) as their 135

CHARACTERS OF GRAM-NEGATIVE BACTERIA

[7.9]

Table 7.9c Third-stage table for the enterobacteria (part 2) 16 Motility Yellow pigment Red pigment M a c C o n k e y growth S i m m o n s ' citrate Christensen's citrate Urease Gelatin hydrolysis Growth in K C N m e d i u m H 2 S (PbAc paper) H2SfromTSI Gluconate Malonate ONPG Phenylalanine Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Selenite reduction Casein hydrolysis D N a s e production Carbohydrates [in Peptone Water m e d i u m ] , gas from glucose acid from: adonitol arabinose cellobiose dulcitol glycerol inositol lactose maltose mannitol raffinose rhamnose salicin sorbitol sucrose trehalose xylose starch M R test (37 °C)a M R test (RT)* 7 V P test (37 °C)a VPtest(RT)^ Indole 16 17 18 19 20 21 22 23

+

17 18

19 20 21

+ — d + _ _ + + _ + _ _

d + + + d _ _ _ _ _ _ _ + + + + — d — — d d + d — — — — — - + d _ _ _ _ _ _ _ _ + d + + + _ _ _ _ d d - + d + d + d + + d + + + _ _ _ _ _ _ _ _

+

d

+ + + + + + + d + + — d + + + +

— + d + d + + d + d + d + + + + _ _ +

_ +

+

+

+

22 23 24 25 26 27 28 29 30

d _ _

_ _

+ + + d + d _

_

_ + d +

_

+ + + _ _

_ _ +

_ _

_ _

_ _

+ + + + d + + + + + + _ — — — — + + d _ _ _ + + + + + + + _ _ _ + + d + + _ _ _ _ _ _ +

+

d

_ _

+ + _ _ _ _ + d + + — + — d — + d + d + - + — — - h - h d d + + d + _ _ _ _ _ _ _ _ + + + + + _ _ + d +d + + d + d _ _ _ d _ _ _ d +

_ _

-

+

+ — — — + + + + + — - + + + + + + + + + + + + + d + + + + + + + d d - + + + + d + d d + + d + + d d d + + d + + + + + + + + + + + + + + + + + + + + + + - + + + + + + + + + + + + + d + + + + d + d + + + + + + — — — d + + + + + d d + + d + + + + + + + + + + + + + + + + + + + + + + + d d d + d + + + d d d + + + + d + + d d + + + + _ _ _ _ d d d _ _ _ d d + d + - + d

+

+ _ _ +

d _ _ +

+ +

d +

+ + + d + + +

+ + d _ + d + +

+

+

+ -

+ + -

— d - — — - + + +

+ + d d d -

+ d + d d -

Escherichia adecarboxylata Escherichia coli Escherichia fergusonii Escherichia hermannii Escherichia vulneris Hafnia alvei; lEnterobacter alveV Klebsiella oxytoca Klebsiella pneumoniae subsp. aerogenes; 'K. aerogenes'; K. pneumoniae (sensu lato); (NOT 'Aerobacter aerogenes'

2 4 Klebsiella p n e u m o n i a e subsp. ozaenae; K. ozaenae 2 5 Klebsiella p n e u m o n i a e subsp. p n e u m o n i a e ; K. pneumoniae (sensu stricto); Friedlander's pneumobacillus 26 Klebsiella pneumoniae subsp. rhinoscleromatis; K. rhinoscleromatis 27 Kluyvera spp. 28 Morganella morganii; 'Proteus morganii'; Morgan's no. 1 bacillus 29 Proteus mirabilis

Beijerinck)

30 Proteus penneri; P. vulgaris biogroup 1

RT, room temperature (18-22 °C);a incubation for two days; ^ incubation for five days Other symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

ENTEROBACTERIA

biotype 32011. Sakazaki (1961) and Ewing (1963) have both suggested that Hafnia should be included in Enterobacter, as 'E. alvei 'and 'E. hafniae', respectively. DNA studies, however, show that H. alvei deserves separate generic status (Brenner, 1978); they also revealed three groups within the species, each of which may eventually be recognized as separate species. As with Serratia, the biochemical characters are subject to temperature variations, and the most typical results are best obtained at 25-30 °C. Minidefinition: Hafnia. Gram-negative rods; motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Attack sugars fermentatively and gas is produced. Gelatin is not liquefied; urea is not hydrolysed. Type species: H. alvei; NCTC strain 8105. 7.9.11 Klebsiella (Tables 7.9a, c). For many years the main problem in identifying Friedlander's bacillus (to which the name K. pneumoniae was later applied) was to distinguish it from lAerobacter aerogenes' (later to be called "K. aerogenes') which was considered to be a non-motile organism. Much less difficult was its separation from the motile organism now known as Enterobacter aerogenes but which for a short time was also known as 'Aerobacter aerogenes' (Hormaeche & Edwards, 1958); by means of a paper-strip test for urease, Matsen (1970) claimed to be able to make the distinction in ten minutes. This contrasts with the lengthy DNA reassociation experiments of Brenner, Steigerwalt & Fanning, (1972) and Brenner (1973) which provided much more convincing (but not routine) evidence for the distinction. Other naming problems now enter the arena since 'Klebsiella aerogenes' (Report, 1956Z?), currently without standing in nomenclature is, by many workers, also named K. pneumoniae. In addition, the subdivision of the klebsiellas in previous editions of this Manual was excessive. The maintenance of 'K atlantae' and 'K. edwardsW as separate species was not supported either by numerical taxonomy of biochemical characters (Bascomb et al., 1971) or by DNA-DNA hybridization studies (Brenner, Steigerwalt & Fanning, 1972), so these two names have now fallen into disuse. The latter study also revealed that organisms appearing in the last edition of this Manual under the names iK. aerogenes', K. ozae-

[7.9] nae, K. pneumoniae (sensu stricto) and K. rhinoscleromatis are, in effect, only biotypes, or subspecies, of a single species with the name K. pneumoniae. What we regarded as K. pneumoniae was a more closely defined taxon than the K. pneumoniae of many other workers (e.g. 0rskov in Bergey's Manual, 1974; Edwards & Ewing, 1972) so we accepted the term used by Bascomb et al. (1971) for our species as K. pneumoniae {sensu stricto) because it helped to contrast with the K. pneumoniae (sensu lato) of 0rskov and others. K. pneumoniae (sensu lato) includes the organisms we previously referred to as 'K. aerogenes', 'K. atlantae' and 'K. edwardsW. Against this scientific background and two differing opinions about the classification of the klebsiellas, it is difficult to draw up a compromise solution. The prevailing view is to accept K. pneumoniae (sensu lato) and reject K. pneumoniae (sensu stricto) as judged by the inclusion of K. pneumoniae in the Approved Lists of Bacterial Names (1980) and the omission of 'K. aerogenes'. In the interests of uniformity we shall accept the name K. pneumoniae for the species as a whole and we foresee that the most widely accepted classification will effectively be K. pneumoniae subspecies pneumoniae (= K. pneumoniae sensu lato), K. pneumoniae subsp. ozaenae (= K. ozaenae) and K. pneumoniae subsp. rhinoscleromatis (= K. rhinoscleromatis). As a temporary measure, we propose amending this classification so that K. pneumoniae subsp. pneumoniae = K. pneumoniae sensu stricto and we shall retain 'K. aerogenes' as a fourth subspecies, K. pneumoniae subsp. aerogenes. We have followed this uneasy compromise in the Tables but it must be remembered that as these four taxa are effectively a single species there are bound to be numerous intermediate forms that do not correspond neatly to any of the taxa. The correlation between the subspecies and capsule serotypes is: K. pneumoniae subsp. pneumoniae (K. pneumoniae sensu stricto), type 3; K. pneumoniae subsp. rhinoscleromatis, type 3; K. pneumoniae subsp. ozaenae, types 3, 4, 5, and 6; and Klebsiella pneumoniae subsp. aerogenes, all types. K. pneumoniae subsp. pneumoniae is associated with acute infections and K. pneumoniae subsp. ozaene with chronic lung disease, but K. pneumoniae subsp. aerogenes is seldom pathogenic (Fallon, 1973). Like most enterobacteria, the reactivity of klebsiella antigens extends beyond the klebsiella group. 137

CHARACTERS OF GRAM-NEGATIVE BACTERIA

Indole-positive forms are probably not uncommon and strains that produce indole and liquefy gelatin have been named K. oxytoca', 0rskov (1955, 1957) and Ewing (1963) prefer to retain them in the genus, but Lautrop (1956&) and Hugh (1959) think that these strains should be excluded from Klebsiella. DNA-DNA relatedness studies suggest, however, that K. oxytoca strains should be placed in a genus separate from Klebsiella (Jain, Radsak & Mannheim, 1974). In Table 7.9c K. oxytoca is included for the indole- and often gelatin-positive forms. Other indole-producing strains have been placed in K. planticola (Bagley, Seidler & Brenner, 1981) and K. trevisanii (Ferragut et al, 1983). Strains of these two species (which may well be synonymous) were isolated from non-clinical sources, and hence are not included in the Tables. However, Freney et al. (1986) reported that 18% of the clinical isolates they examined and which had been identified initially as K. oxytoca, proved to be strains of K. trevisanii. Some strains of Klebsiella pneumoniae (sensu lato) are able to fix atmospheric nitrogen (Mahl et al., 1965) but this character does not seem to have any known taxonomic value and cannot be used in the identification of Klebsiella species. Minidefinition: Klebsiella. Gram-negative rods; non-motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Attack sugars fermentatively, usually with the production of gas. KCN- and VPpositive (important exceptions). Ornithine decarboxylase not produced. Urea generally hydrolysed. Phenylalanine-negative. Type species: K. pneumoniae; NCTC strain 9633. 7.9.12 Kluyvera (Tables 7.9a, c). This genus was proposed by Asai et al. (1956) with two species, 'K. citrophila\ to accommodate citrate-utilizing strains and 'K. noncitrophild* for strains not utilizing citrate, the principal feature differentiating them from Escherichia being their apparent polar flagella. In 1962, Asai et al. published evidence showing that their strains were peritrichously flagellated and they proposed transferring them to the genus Escherichia. These species names, which had been little used, were not included in the Approved Lists of Bacterial Names (1980). However, the generic name Kluyvera was revived by Farmer et al. (1981) with two species, 138

[7.9]

K. ascorbata (whose strains gave a positive result in the ascorbate test and failed to produce acid from glucose after incubation for 21 days at 5 °C) and K. cryocrescens (whose strains gave the opposite results in the above tests). As it is impracticable to differentiate between the two species routinely by biochemical tests we combine them in the Tables as a single taxon Kluyvera spp. However, strains of K cryocrescens generally display large zones of inhibition around discs of carbenicillin and cephalothin whereas strains of K. ascorbata fail to do so. Kluyvera strains can easily be confused with strains of Buttiauxella and Citrobacter. Most of the reported strains have been isolated from sputum and smaller numbers from urine, faeces and blood cultures; diarrhoea possibly caused by Kluyvera species has also been reported (Fainstein et al, 1982; Aevaliotis et al, 1985). Minidefinition: Kluyvera. Gram-negative rods; motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Attack sugars fermentatively; gas is produced. Sucrose-positive. Generally citrate-positive; generally KCN-positive. Arginine-negative. Type species: K. ascorbata; ATCC strain 33433. 7.9.13. Morganella (Tables 7.9a, c). Bacteriologists have for long been fascinated by Morgan's number 1 bacillus (Morgan, 1906), a quite unimportant organism in spite of his belief that it was the cause of 'summer diarrhoea' in infants. Thjotta (1920) thought that it was a 'metacolon' (Escherichia coli in today's terms); Jordan, Crawford & McBroom, (1935) doubted whether there was a species well enough characterized to be recognized and preferred to think of it as a large and variable group. To Rauss (1936) it was a member of the Proteus group, and he showed that on a solid medium of low agar content it would swarm as Proteus did on ordinary Nutrient Agar media. Fulton (1943) proposed a separate genus, Morganella, for it. Richard (1965) found that it had four to ten times the urease activity of other Proteus species, and he thought that this put it into a category of its own. Meanwhile, Rauss & Voros (1959) had joined the ranks of the separatists and supported the creation of Morganella. Convincing evidence for a difference between

ENTEROBACTERIA

Morganella morganii and Proteus species has come from research. The urease and phenylalanine deaminase enzymes are said to be serologically distinct from those of Proteus species; and the G+C content of the DNA for Morganella morganii is 50% and for Proteus species 40% (Penner, 1984a,fr). Such a wide difference is unlikely to be found between members of the same genus. Unfortunately this kind of information is obtained by methods that are not yet available in routine clinical laboratories; we must make the distinction and identification from the results of routine tests and include sufficient characters in the tables to show up differences in as convincing a manner as possible. From a strictly medical viewpoint, it is doubtful if the effort to distinguish these organisms is worthwhile, for neither Morganella nor Proteus species appear to be convincing pathogens though as laboratory nuisances they both hold high places. Minidefinition: Morganella. Gram-negative rods; motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Glucose is fermented with the production of a small bubble of gas. Phenylalaninepositive; urease-positive. Citrate-negative; gelatin-negative. Type species: M. morganii; NCTC strain 235. 7.9.14 Proteus (Tables 7.9a, c, d). The collection of species in the genera Proteus and Providencia and the various proposals to combine them and then to separate them again have the monotonous sounds of much taxonomic argument; we shall summarize this and give references for those who wish to pursue it. Rustigian & Stuart (1945) proposed that four species of the genus Proteus should be recognized, P. vulgaris, P. mirabilis, P. morganii, and P. rettgeri', this arrangement gained wide acceptance, though a few preferred to combine the first two species as "Proteus hauserV. Because the '29911 group' of organisms of Stuart, Wheeler & McGann, (1946) was phenylalanine-positive it was added to Proteus by Singer & Bar-Chay (1954), Buttiaux et al. (1954) and Shaw & Clarke (1955) as the 'Providence group' or as Proteus inconstans. In the opposite direction, several splittings and new genera were proposed: Morganella by Fulton (1943) for Morgan's No. 1 bacillus; Proom & Woiwod (1951) suggested the exclusion of Rettger's bacillus

[7.9] from Proteus, and Kauffmann (1953) created a new genus for it CRettgerella'). Kauffmann & Edwards (1952) listed a new genus Providencia; Ewing (1962) described it and named two species, P. alcalifaciens and P. stuartii. Rauss & Voros (1959) and Rauss (1962) supported the proposals for new genera {Morganella, LRettgerella\ and Providencia) but Richard (1966) thought that Rettger's bacillus should be included in Providencia. Lautrop, in Bergey's Manual (1974), reviewed the situation as it existed early in the 1970s and discussed the possible taxonomic treatments. His general conclusion was that Morgan's bacillus, with its G+C content of DNA of 50%, compared with 40% for the other Proteus species, seemed the best candidate for separate recognition. Since then, DNA-DNA hybridization studies have confirmed that Morganella morganii deserves separate generic status as it is more closely related to other Enterobacteriaceae than to Proteus or Providencia (Brenner et al., 1978). These studies also confirmed that the two biotypes A and B of Proteus inconstans deserve separate generic status and should also be classified as separate species: Providencia alcalifaciens and Providencia stuartii respectively. Despite its ability to produce urease, which served as a useful test to distinguish Proteus and Providencia, DNA studies also showed that Proteus rettgeri should be transferred to Providencia as Providencia rettgeri leaving only P. vulgaris and P. mirabilis as distinct species in the genus Proteus. However, more detailed DNA-DNA hybridization studies revealed that P. vulgaris can be subdivided into three biogroups, each of which should probably be recognized as separate species (Hickman et al., 1982). Biogroup 1 strains do not produce indole and fail to ferment salicin (the name Proteus penneri has been proposed for them); biogroup 2 strains produce indole and ferment salicin; biogroup 3 strains produce indole but do not ferment salicin. The type strain of P. vulgaris belongs to biogroup 3 so this name may ultimately be reserved for strains of biogroup 3 and yet another new name may be proposed for biogroup 2. We have followed this current classification in the Tables. The usual biochemical characterizing tests often reveal aberrant strains as, for example, the indole-positive Proteus mirabilis strains reported by Matsen et al. (1972), and lactose-fermenting strains of Providencia rettgeri (Sutter & Foecking, 1962). 139

CHARACTERS OF GRAM-NEGATIVE BACTERIA

In the medical laboratory, Proteus species, because of their swarming property, are a nuisance and rarely a subject for identification. Various tricks have been devised to inhibit swarming: the inclusion of bile salts in the medium (as in MacConkey Agar) is one of the most successful, though it may make the medium unsuitable for the growth of the organisms we want to isolate. Other measures include exposure to ether vapour (Bray, 1945), chloral hydrate (Gillespie, 1948); increased concentrations of agar (Hayward & Miles, 1943) or NaCl (Kopper, 1962); and deletion of added salts as in the Cystine-LactoseElectrolyte Deficient (CLED) medium of Mackey & Sandys (1966). We have not yet mentioned Proteus OX strains; these are strains of Proteus vulgaris which form the basis of the Weil-Felix reactions in patients with typhus infections. Such strains do not need to be identified among laboratory isolates of P. vulgaris from clinical material; their importance lies solely in their use as diagnostic agents. Although they are not connected in any way with the infecting agents (Rickettsia prowazekii or R. typhi), they are, in the O (non-motile) form, agglutinated by the sera of typhus patients, a titre of 1 in 50 or more suggesting a rickettsial infection which should be confirmed by a rise in titre during the course of the clinical illness. Proteus 0X19 is agglutinated by sera from patients with classical typhus (R. prowazekii infection) and Proteus OXK by sera of patients with tsutsugamushi fever (R tsutsugamushi infection). These tests, however, are likely to become less useful in future with the application of specific sero-diagnostic methods. Minidefinition: Proteus. Gram-negative rods; motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Attack sugars fermentatively, usually with gas production. Phenylalanine-positive; urea hydrolysed. Gelatin hydrolysed. Type species: P. vulgaris; NCTC strain 4175. 7.9.15 Providencia (Tables 7.9a, d). The history of the genera Morganella, Proteus and Providencia is so inextricably interlinked that, having already covered Morganella and Proteus in detail, there now remains little further to add on Providencia. Although three species, P. alcalifaciens, P. rettgeri 140

[7.9]

and P. stuartii, are generally recognised and are included in the Tables, DNA-DNA hybridization data show that P. alcalifaciens can be further subdivided (Hickman-Brenner et al., 1983), so that strains fermenting galactose though not adonitol should now be classified as Providencia rustigianii. Minidefinition: Providencia. Gram-negative rods; motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Attack sugars fermentatively, generally without gas production. Phenylalanine-positive; gelatin not hydrolysed. Type species: P. alcalifaciens; NCTC strain 10286. 7.9.16 Salmonella (Tables 7.9a, d). The identification of a bacterium as a salmonella is not difficult but, with that done, two problems arise. The first is the complex antigenic analysis and often phage typing needed to identify strains in sufficient detail to be helpful in tracing the source of infection; the second is how to label (by name or antigenic formula) the strain in the report that must be made to the clinician and to public health officials. The Salmonella Subcommittee (1934) thought that serology was the ultimate criterion in the classification (and, by implication, identification) of the group though they did not suggest that it should be based on serology alone. Complete antigenic analysis and phage typing are not routine procedures for clinical laboratories, but reference laboratories are available in most countries. Simplified diagnostic schemes using a limited number of antisera are usually used (Edwards & Kauffman, 1952; Spicer, 1956) permitting the presumptive serotyping of many salmonellas isolated from clinical material. It should be noted that many of the serotypes are also known by well-established names, which have no formal standing in nomenclature, such as 'S. pullorum\ 'S. gallinarum' and 'S. paratyphi A \ A polyvalent salmonella phage was of some use in distinguishing salmonellas from other enterobacteria but it was not a substitute for classical methods (Talley, 1968). More recently, DNA hybridization methods have been described to detect salmonellas in foods (Fitts et al., 1983) and to identify Salmonella typhi in stools (Rubin et al., 1985); such probes are likely to become widely used in the future. One serotype, S. typhi, is distinct both serological-

ENTEROBACTERIA

ly and biochemically and warrants separate description; it differs from most other salmonellas in being citrate negative (Simmons' medium), in not producing gas from glucose and in producing only small amounts of H2S. 'S.gallinarum\ which causes disease in poultry, does not produce gas and other serotypes may occasionally yield anaerogenic variants. 'S. paratyphi A' is also not typical in several characters: few strains produce H2S from TSI slopes; it does not use citrate as a carbon source or produce acid from xylose; and it does not decarboxylate lysine. In addition, the type species of the genus, Salmonella choleraesuis, is biochemically atypical and does not ferment arabinose or trehalose. Lactosefermenting strains that are otherwise biochemically typical are rare but do occur among several serotypes (Threlfall, Hall & Rowe, 1983). Members of the Arizona group (S. arizonae) often ferment lactose although this reaction may be delayed; in addition they are ONPG-positive, liquefy gelatin and are usually malonate-positive (Shaw, 1956). Most other Salmonella serotypes are biochemical variants with a fairly stable pattern, and Borman, Stuart & Wheeler (1944) proposed that they should all be called 'Salmonella kauffmannif'. For a similar all-embracing species Kauffmann & Edwards (1952) suggested the name 'Salmonella enterica\ which would have been unusually meaningful and has recently found favour (see below); Ewing (1963) proposed that the name Salmonella enteritidis should be followed by the serotype name (when it had one) or the antigenic formula; this system has been tried in the USA but failed to gain acceptance elsewhere. Kauffmann (1963a) divided the Salmonella group into what he called sub-genera; these are now widely accepted as distinct groups (though not as subgenera) and we therefore include them in Table 1.9d. If Salmonella is regarded as a genus, then these groups could conveniently be considered as species: 'Salmonella kaujfmanniV (subgenus I), 'S. salamae' (II), S. arizonae (III), and 'S. houtenae' (IV) (Le Minor, Rohde & Taylor, 1970). Later Le Minor, Veron & Popoff (1982) proposed on the basis of numerical taxonomy and DNA relatedness that the genus Salmonella should consist of a single species composed of six subspecies. S. choleraesuis was proposed as the type species with the six subspecies as follows: subsp. choleraesuis would include all sub-

[7.9] genus I strains; subsp. salamae would correspond to subgenus II; subsp. arizonae would include the monophasic serotypes of subgenus III while subsp. diarizonae would include the diphasic serotypes; subsp. houtenae would correspond to subgenus IV and subsp. bongori would consist of ONPG- and KCN-positive strains that fermented dulcitol. A seventh subspecies, indica, has since been proposed for strains that are gelatinase-positive but negative for malonate, tartrate, salicin and sorbitol (Le Minor et ah, 1986). More recently, a new proposal to change the type species to 'S. enterica' has been published (Le Minor & Popoff, 1987); this proposal has the advantage that the name has not been used previously for any bioserovar of Salmonella and it is thus less likely to cause confusion. It remains to be seen whether these latest proposals gain wide acceptance. We are sceptical about these proposals and think that, for practical purposes, clinical laboratories are likely to continue to use serotype names such as S. typhimurium rather than the cumbersome equivalent 'S. choleraesuis [ or S. enterica] subsp. choleraesuis bioserovar Typhimurium'. The typically non-motile salmonellas {'S. gallinarum' and 'S. pullorum') from poultry are often combined, but as they differ from each other in several characters we show them separately in Table 1.9d. Although numerous isolation media are described, they do not seem to have changed much in principle since Hobbs & Allison (1945), after comparing several media, found that of Wilson & Blair (1931) to be the most valuable solid medium with Deoxycholate Citrate Agar a close second; they recommended, however, that both media should be used as the combination gave more Salmonella isolations than either medium alone. For enrichment, Selenite F (F for faeces) gave the best results when plated on Wilson & Blair's Bismuth Sulphite agar medium. Harvey & Thomson (1953) found that cultures incubated at 43 °C yielded more positive results but temperature regulation had to be accurate as 44 °C was inhibitory for salmonellas. Selenite F incubated at 43 °C is also recommended by Georgala & Boothroyd (1965) followed by plating on Brilliant Green Agar. For a summary of available media, see Ewing (1986) and Fricker(1987). Minidefinition: Salmonella. Gram-negative rods; motile (a few exceptions). Aerobic and 141

[7.9]

CHARACTERS OF GRAM-NEGATIVE BACTERIA

Table 7.9*/. Third-stage table for the enterobacteria (part 3) 31 Motility Yellow pigment Red pigment MacConkey growth Simmons' citrate Christensen's citrate Urease Gelatin hydrolysis Growth in KCN medium H 2 S (PbAc paper) H 2 SfromTSI Gluconate Malonate ONPG Phenylalanine Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Selenite reduction Casein hydrolysis DNase production Carbohydrates [in Peptone Water medium], gas from glucose acid from: adonitol arabinose cellobiose dulcitol glycerol inositol lactose maltose mannitol raffinose rhamnose salicin sorbitol sucrose trehalose xylose starch MR test (37 °C)a MR test (RT)^ VP test (37 °CT VPtest(RT)^ Indole

d + d + + + + d _ _ + + + + _ + + + d + + + +

32

33

d d _ _ _ _ + + - d d + + _ + d + + + d d _ _ _ _ _ _ + + d d d _ + +

34 _ _

+

_ _

+ + + + + +

d

36 +

_ _

+ + +

+ d + _ -

-

+ d d d _ _ _ _ _ _ _ _ _ + + + + + + d d _ _ _ d d d

d

_ d _ _ + d _ _ + _ _ + _ + + + _ _ + + + + _ _ _ _ + +

35

-

-

_

_

+

_ _ d _

+

-

-

_ _ d

-

_ _

+ + _ _ +

31 Proteus vulgaris biogroup 2 32 Proteus vulgaris biogroup 3 33 Providencia alcalifaciens; Proteus inconstans; 'Proteus inconstans A'; Proteus providenciae'; 'Providencia providenciae' 34 Providencia rettgeri; 'Proteus rettgerV; 'Rettgerella rettgerV 35 Providencia stuartii; 'Proteus stuartiV; 'Proteus inconstans B'; ' Proteus providenciae B' 36 Salmonella choleraesuis 37 ' Salmonella gallinarum'

_

-

_ + + _ _ +

_

d + d

_ _

d + d _

+ +

_ _ — —

42

43

-

+

+ _ _ + + + _ d + + _ +

+ _ _ + + + _ d

+ _ _ + + + _ d + + + _

_ _

+ +

_

d -

_ _ _ _ d + + +

+ _

_

+ +

+ + + + _ _ d d

_

_ -

+

+ + + + _ d

+ + _ + + + + + + _ d

+

+

+

-

d

_ + -

d d _

d

_ + d d + + _ + d + + + _ + + _ _

_ d d + + _ + + + _ + + _ _

_ +

_

_ +

d +

+ +

d d _

44

+ _ _ + + _ + d _ _ _ - + + + + + + + _ _ -

d

+ d -

_

-

_ +

_ + +

d

+

_ _

+ + +

+

+

_

+ d +

d +

41

+

+

_

+ d -

_

40

_ -

-

_

39

-

_ + d d d d _ _ _ _ _ _ _ _ _ + + + d + _ _ _ d d -

+

d

_

+

_

+ +

_ _

+

+ _

d + +

+

_ _ -

_ -

-

-

38

+ -

_ -

d

-

_ _

+

+ -

37

+

-

+ + + + _ _ _ _ _ _ + d + + + + + + + + + + + + - d + + + _ _ _ _ _ _ + + + + + + + + + + _ _ _ _ _ _ _ _ _ _ _ _ — — — — — -

38 'Salmonella paratyphi A' 39 'Salmonella pullorum' '40 Salmonella subgenus I; 'S. kauffmannii'; 'S. enterica'; S. enteritidis serotype (bioser) xyz 41 Salmonella subgenus II; 'S. salamae'; 'S. dar-es-salaam' 42 Salmonella subgenus III; 'Arizona arizonae'; ' A. hinshawii'; Salmonella arizonae 43 Salmonella subgenus IV; 'S. houtenae' 44 Salmonella typhi

RT, room temperature (18-22 °C);a incubation for two days; b incubation for five days Other symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

ENTEROBACTERIA

facultatively anaerobic. Catalase-positive; oxidase-negative. Attack sugars by fermentation with production of gas. Simmons' citrate usually positive (S. typhi is an important exception that does not produce gas and is Simmons' citrate-negative). KCN-negative {except subgenus IV); lysine decarboxylase usually positive (with the exception of 'S. paratyphi A'). Type species: S. choleraesuis; NCTC strain 5735. 7.9.17 Serratia (Tables 1.9a,e). Classically, members of certain species of this genus produce a bright pink or red pigment though in culture non-pigmented variants are constantly thrown off; non-pigmented strains occur also in nature and, in some species, all strains are non-pigmented. In a five month period, 92% of Serratia strains isolated in a Boston hospital were not pigmented (Wilfert et al, 1970); consequently, bacteriologists must be able to identify this organism in the absence of pigment. However, if pigment is present, identification is much easier, and various media have therefore been devised to encourage its production. Meat extracts seem to inhibit pigment formation but it can be restored by transfer to a medium without meat (Goldsworthy & Still, 1936). Kharasch, Conway & Bloom, (1936) thought that glucose was necessary for pigment production by Serratia marcescens; on the other hand, Goldsworthy & Still (1936, 1938) found that glucose inhibited and mannitol stimulated pigment production by their strains. Cowan & Steel (1974) confirmed these findings, which indicate that strains vary in the way in which they respond to glucose in the medium. One of the most satisfactory media for showing pigment potential is medium A of King, Ward & Raney, (1954) (Appendix A2.4.3); Sedlak and his colleagues (1965) reported good results with Dorset Egg medium at 22 C. As with Hafnia, the most characteristic results of biochemical tests are obtained at about 25 °C. Sometimes the occurrence of serratias in human secretions can be misleading for the unwary; for example, their growth in sputum after expectoration may suggest haemoptysis (Robinson & Woolley, 1957). Serratias may cause occasional infections, especially in children and, with the recognition of non-pigmented strains, reports of their isolation have

[7.9] become more frequent (see, for example, Brooks, Chambers & Tabaqchali, 1979). Serratia marcescens liquefies gelatin rapidly and, in this and other characters, Pederson & Breed (1928) noticed that it closely resembled the liquefying species of ''Aerobactef (subsequently 'Enterobacter liquefaciens') and thought that such non-pigmented strains might be included in Serratia. The resemblance extends to deoxyribonuclease activity (DNase) which occurs more frequently and is more active in strains of Serratia marcescens and 'Enterobacter liquefaciens' than in most other enterobacteria (Voros, 1969). Black, Hodgson & McKechnie, (1971) compared methods for carrying out this test in routine laboratories and preferred that of Jeffries, Holtman & Guse, (1957). A rapid and easily read method (combined with the PPA test) was described by Oberhofer & Maddox (1970). The suggestion of Pederson & Breed (1928), which has been repeated by many others, has been generally accepted and '£. liquefaciens' is now included in Serratia as S. liquefaciens. Serratia marcescens can be typed for epidemiological purposes by their O and H antigen serology (Traub, 1985), by bacteriocins (Anderhub et al., 1977; Traub, 1980), bacteriophages (Pitt, Erdman & Bucher, 1980), and by biotyping (see Pitt, 1982). Biotyping of S. marcescens was described by Grimont & Grimont (1978a,Z?) who tested strains for the ability to utilise various substrates. Conventional tests used for species identification are not generally suitable for biotyping due to the similarity of biochemical reactions within the species. Another species, Serratia rubidaea, described by Breed in Bergey's Manual (1948) as having characters much like those of S. marcescens, has been revived by Ewing, Davis & Fife, (1972); although some strains came from human sources the clinical significance was not indicated and presumably was unknown. In several instances the strains described by Ewing and his colleagues, and for which an old name was revived, differ in their characteristics from those on which the original description of the species was based. S. rubidaea is no exception and we prefer to use the name S. marinorubra for this organism (see Grimont et al, 1977), which is anaerogenic and does not decarboxylate ornithine. Serratia odorifera was described from 25 strains isolated mostly from clinical material; of these 17 were designated biotype 1 143

[7.9]

CHARACTERS OF GRAM-NEGATIVE BACTERIA

Table 1.9e. Third-stage table for the enterobacteria (part 4)

Motility Yellow pigment Red pigment MacConkey growth Simmons' citrate Christensen's citrate Urease Gelatin hydrolysis Growth in KCN medium H 2 S (PbAc paper) H2SfromTSI Gluconate Malonate ONPG Phenylalanine Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Selenite reduction Casein hydrolysis DNase production Carbohydrates [in Peptone Water medium], gas from glucose acid from: adonitol arabinose cellobiose dulcitol glycerol inositol lactose maltose mannitol raffinose rhamnose salicin sorbitol sucrose trehalose xylose starch M R test (37 °C)a MRtest(RT)^ V P test (37 °C)a VPtest(RT)^ Indole 45

45

46

47

+

+ _

+ _

— + + + + +

—

_ + + d d d

+ + + d + + _ _ + _ + _ _ + + + + +

d

d

d +

_ < j + d _ + + + + + + + + + + + + + + + + + + _ + _ _ _ + d + _

d

48C + _ —

-

+ d d _ + _ + _ _ d + d

+ + _ + d + _ _

_ + + d + + + + + + + + + _ +

+ + + + _ d _

_

_ d d _

+

-

_ d +

— +

_

_ -

-

-

+ _ _ _ _ + d d + d + d + + + + + + d d + + + + + d + + + + + + _ _ _ + d + _ d + d d _ _ _ _

Serratia liquefaciens; 'Enterobacter liquefaciens'; ' Aerobacter liquefaciens' '46 Serratia marcescens; 'Erythrobacillus prodigiosus'; ' Chromobacterium prodigiosum' 47 Serratia marinorubra; S rubidaea, Serratia biotype II (Bascomb etal, 1971); Serratia Phenon B (Grimont et al, 1977) 48C Serratia odorifera biovar I 49 Serratia odorifera biovar II 50 Serratia plymuthica 51 Shigella sonnei

d (

_ — +

— -

—

d -

-

-

—

+ +

j

+

d -

-

-

_ -

— +

—

-

_ — _

-

-

-

+ _

d -

—

+ d

— -

+ + d d

_ -

— _

d

—

-

d

-

_

-

_

d + d

56

— + d + +

—

-

d

-

+

_ — +

+ — +

-

_

d

55

+

_ -

d _

54C _ —

-

-

d + + +

—

-

+ d

53

_ —

_ — +

-

_

52<* -

_ — + d +

-

d + +

-

_

+ + + d

d

+ + + + + +

-

_

-

_

+

+ + d _ + + _

+ + d

+ + +

+

-

d -

51

d + + +

+ + +

-

50

_ —

+ + +

49

_

_ + d d + + + + + + + + +

+ + + + + + _ + +

+

_

_ d d + -

-

+ + +

+ -

_ + +

d +

-

+

d + +

59

-

+ +

_ +

58

—

+ +

_

d + d d d d + d + d d + + + + + + d _ _ _ _ + + + + d d d d

—

+ +

57

+ + d _ _ _ _ — — + + + d + + + + + + + _ _ _ _ — + + + + _ _ _ _ — — + + d d d -

d +

+ + _ + + d

+ + _ d + -

+ _ + + + + d + + _ + + -

52^ Shigella spp. (excluding S. sonnei); S. boydii ([serotypes] 1-15; Boyd's dysentert bacilli); S. dysenteriae ([serotype 1: 'S. shigae'; Shiga's bacillus]; [serotypes 2-10; Large-Sach's group; 2 = 'S. schmitziV; 'S. ambigua'; Schmitz's bacillus]); S.flexneri ([serotypes 1-5: Flexner's dysentery bacilli]; [serotype 6: Boyd 88]; Manchester bacillus; see Table 7.9/) 53 Tatumella ptyseos C 54 Yersinia enterocoliticia; 'Pasteurella X' 55 Yersinia fredericksenii 56 Yersinia intermedia 57 Yersinia kristensenii 58 Yersinia pestis; 'Pasteurella pestis'; the plague bacillus 59 Yersinia pseudotuberculosis; 'Pasteurella pseudotuberculosis'; P. rodentium'

RT, room temperature (18-22 °C);a incubation for two days; b incubation for five days; c Some strains nitrate-negative; d Some strains catalase-negative. Other symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

ENTEROBACTERIA

and the remaining eight as biotype 2 (Grimont et al, 1978). Strains of biotype 1 decarboxylate ornithine, and ferment raffinose and sucrose whereas those of biotype 2 give the converse results in these tests; their clinical significance is unknown. Other species of Serratia reported occasionally in clinical material include S. ficaria (normally associated with figs and fig wasps), from respiratory specimens (Gill et al, 1981; Brouillard, Hansen & Compete, 1984) and from a leg ulcer (Pien & Farmer, 1983), as well as 5. plymuthica from a burn site (Clark & Janda, 1985). S. fonticola, isolated from water, was described by Gavini et al. (1979) but, after numerical analysis of the genus, Feltham & Stevens (1983) queried its inclusion; we do not therefore show this environmental organism in the tables. Minidefinition: Serratia. Gram-negative rods; motile. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Attack sugars fermentatively, often with gas production in some species. VP usually positive. Gluconate-positive. Strains of some species produce ornithine decarboxylase; deoxyribonuclease-positive. Strains of some species produce a red pigment when grown on suitable media. Type species: S. marcescens; NCTC strain 10211. 7.9.18 Shigella (Tables 7.9a, e, f). Modern ideas on the classification of this genus owe much to Ewing, who built on the foundations laid by Murray (1918), Andrewes & Inman (1919), and by Boyd (1938) whose recognition of specific and group antigens introduced some system into the serological analysis of shigellas. Ewing (1953) also showed that many strains previously thought to be shigellas are best regarded as non-motile, anaerogenic strains of E. coli. More recently, DNA hybridization studies have confirmed that Shigella and E. coli belong to a single genetic species (Brenner et al, 1982c). It is therefore not surprising that most shigellas are closely related to O groups of E. coli (Edwards & Ewing, 1972; Rowe, Gross & Guiney, 1976). The distinction between Shigella and E. coli in the diagnostic laboratory depends on only a few tests and those for motility, lysine decarboxylase and the utilization of citrate

[7.9] (Christensen's medium) are of particular value. Biochemically the Shigella group is divided into the mannitol non-fermenters (Shigella dysenteriae) and the mannitol fermenters (S. boydii, S. flexneri and S. sonnei) although exceptions occur with most serotypes. Shigella dysenteriae serotype 1 (Shiga's bacillus) differs from all the others in that it is catalase-negative; among other shigellas catalase-negative strains occur occasionally, especially with S. flexneri type 4a (Carpenter & Lachowicz, 1959), but it is not a constant character of any serotype except S. dysenteriae 1. Strains of S. dysenteriae 2 (Schmitz's bacillus) always produce indole, a character shared among the mannitol non-fermenters by the rarer serotypes 7 and 8. Among the mannitol fermenters the distinction between Shigella flexneri (subgroup B) and S. boydii (subgroup C) is made entirely on serological grounds; strains of S. flexneri possess common group antigens that do not occur in the other subgroups. Strains of Shigella flexneri 6 may differ in some biochemical and immunological characters from serotypes 1 to 5, and Russian workers believe that S. flexneri 6 should be transferred to the S. boydii subgroup (Petrovskaya & Khomenko, 1979); the special characteristics of the Manchester and Newcastle varieties (Downie, Wade & Young, 1933) of S. flexneri 6 are shown in Table 7.9/, which is adapted from that of Carpenter, Lapage & Steel, (1966). It should be noted that some strains do produce gas. Shigella sonnei (subgroup D) produces colicines which, when tested on 'indicator' strains, can be used to sub-divide strains into epidemiologically significant types (Abbot & Shannon, 1958). Typically S. sonnei is indole-negative and most (though not all) strains are late fermenters of lactose and sucrose; Szturm-Rubinsten (1963) recognized different biotypes. The ONPG test confirms their inherent ability to ferment lactose (Le Minor & Ben Hamida, 1962) and with other shigellas many serotypes (especially S. dysenteriae 1) also give positive results in this test (Szturm-Rubinsten & Piechaud, 1963). The antigenic relationship betweeen S. sonnei and Plesiomonas shigelloides should be borne in mind (Ferguson & Henderson, 1947; Martin, Mock & Ewing, 1968).

145

CHARACTERS OF GRAM-NEGATIVE BACTERIA

Table 7.9/. Unusual biochemical characteristics of biotypes 0/Shigella flexneri 6 (modified from Carpenter et al., 1966)

Glucose (acid) Glucose (gas) Mannitol (acid) Dulcitol (acid) Indole

+ + d

+ + (d) -

+ + + (d) -

+ d (d) -

1 Shigella flexneri serotypes 1-5 2 Shigella flexneri 6; Boyd 88 biotype 3 Shigella flexneri 6; Manchester biotype; Denton strains (Downier al, 1933) 4 Shigella flexneri 6; Newcastle biotype; 'Shigella new castle' Symbols used in the table are explained in Tables 5.1 and 5.2 on pp. 47.

For the isolation of shigellas it is preferable to culture freshly passed stools, although if this is not possible rectal swabs showing marked faecal staining may be used. Ideally specimens should be taken during the acute stage of illness before any chemotherapy is given and examined as soon as possible after collection. Enrichment with 'Gram-negative (GN) broth' (Hajna, 1955) may be advantageous but isolation is normally possible by direct plating, especially if blood and mucus is present. Some strains grow poorly on inhibitory media and it is therefore advisable to use a relatively non-inhibitory medium, such as MacConkey or Eosin Methylene Blue (EMB) agar, as well as an inhibitory medium, such as Deoxycholate Citrate Agar (DCA) or Salmonella-Shigella (SS) agar. After overnight incubation at 37 °C, non-lactose-fermenting colonies are selected for further examination, but it should be noted that there may be only a scanty growth of Shigella, even with fresh specimens taken during the acute stage of diarrhoea. In preliminary evaluation, a new selective medium containing potassium tellurite performed better than MacConkey and SS agar used together (Rahaman et al., 1986). Minidefinition: Shigella. Gram-negative rods; non-motile. Aerobic and facultatively anaerobic. Catalase-positive (one important exception); oxidase-negative. Attack sugars by fermentation without gas production (a few exceptions produce gas). Citrate-nega146

[7.9] tive; KCN-negative; lysine decarboxylasenegative. Type species: S. dysenteriae; NCTC strain 4837.

7.9.19 Tatumella (Tables 7.9a, e). There is but a single species, T. ptyseos, strains of which have been isolated mostly from sputa (Hollis et al., 1981). The organism is unusual among the Enterobacteriaceae in having a small number of flagella (usually only one), in displaying a large zone of inhibition around penicillin discs, and in surviving for only a comparatively short period on some laboratory media. Growth occurs on MacConkey agar and most strains are reported to be motile and to utilize citrate (Simmons' medium) at 25 °C but give negative results in these tests at 35-37 °C. They produce phenylalanine deaminase weakly in a suitable medium. Their clinical significance is uncertain. Minidefinition: Tatumella. Gram-negative rods; motile, but with few (usually only one) flagella. Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Attack (fermentatively) only glucose, salicin, sucrose and trehalose; gas is not produced. Type species: T. ptyseos; NCTC strain 11468. 7.9.20 Yersinia (Tables 7.7; 7.9a, e, g) is a genus originally created for the plague and pseudotuberculosis organisms by van Loghem (1944-5), who separated them from the pasteurellas. Other organisms have since been added: Yersinia enterocolitica (Pasteurella X) isolated from animals and man (Mollaret & Chevalier, 1964) and Yersinia ruckeri from rainbow trout with red mouth disease (Ewing et al., 1978). As a result of further studies on Y. enterocolitica and similar organisms described as Y. enterocolitica-like, Yersinia enterocolitica (sensu stricto) was redefined by Bercovier et al. (1980a) and three new species isolated from the aquatic environment and from man were described: Y. frederiksenii (Ursing et al., 1980), Y. intermedia (Brenner et al., 1980a) and Y. kristensenii (Bercovier et al, 1980£). Subsequently, four further species were described: Y. aldovae from fresh water (Bercovier et al., 1984), Y. rohdei from human and dog faeces and surface water (Aleksic) et al., 1987); and Y. mollaretii and Y. bercovieri from human stools and other sources

[7.9]

ENTEROBACTERIA

Table 7.9g. Third-stage table for Yersinia 10 Motility at 37 °C Motility at 20 °C Simmons' citrate Nitrate reduced Glucose, gas from Carbohydrates, acid from arabinose cellobiose fucose lactose maltose melibiose raffinose rhamnose salicin sorbitol sorbose sucrose trehalose xylose Mucate ONPG Pyrazinamidase Aesculin Indole Urease Lipase at 22 °C Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase VP at 22-30 °C 1 2 3 4 5 6 7 8

+ + _b

+ + d d + d _ d + d + +

(+) + d d + + + + +

+ + -

+ + -

+ d -

+ -

+ (d) + -

+ + d

+ + d + _ + d + + (+) d d + +

+ + d d + d + + d d _ + + +

+ + _ + _ d d d _ _ _ + d d

+ + +

+ _ + d + + (+) + d _

(+) + + +

Yersinia enterocolitica biovar 1 Yersinia enterocolitica biovar 2 Yersinia enterocolitica biovar 3 Yersinia enterocolitica biovar 4 Yersinia enterocolitica biovar 5 Yersinia frederiksenii Yersinia intermedia Yersinia kristensenii

+ (d) + -

+ d + +

(d) + -

(+) (+) + + + + +

(+) + + + + + (+) d + + + + + d + +

(+) d + + + d + d + +

9 10 11 12 13 14 15

+ + -

+ (d + -

+ + d (d) + _

+ d

(d) + + + (+) + + (d) d + d + _

+ + + + (+ d + +

+ d +

11

12

13

14

15

+

+

+

+

+

+

(d)

(d)

(d)

Yersinia aldovae Yersinia bercovieri; Y. enterocolitica biogroup 3B Yersinia mollaretti; Y. enterocolitica biogroup 3A Yersinia rohdei Yersinia pseudotuberculosis; 'Pasteurella pseudotuberculosis' Yersinia pestis; 'Pasteurellapestis'; plague bacillus Yersinia ruckeri; red mouth bacterium

a Positive only at 22-25 °C. b A few strains produce a small amount of gas. Other symbols used in the Table are explained in Tables 5.1 and 5.2 on p47.

(Wauters et al., 1988). The so-called *Yersinia philomiragia' (Jensen, Owen & Jellison, 1969) was shown not to be a member of Yersinia or indeed of the Enterobacteriaceae (Ursing, Steigerwalt & Brenner, 1980a) but probably belongs to the genus Francisella. We include Yersinia in Table 7.7 for comparison with Pasteurella and in Tables 1.9a,e for comparison with the other enterobacteria; we give the characters of Yersinia species in Table 7.9g.

It is worth noting that the temperature of incubation is important in tests for the characterization of yersinias. Y. pestis produces a capsule (or envelope) at 37 °C but not at 20-22 °C (Schutze, 1932a); Y pseudotuberculosis is motile at 22 C but not at 37 C (Preston & Maitland, 1952); and while Y. enterocolitica is VP negative at 37 °C, it may be positive at 25-30 °C. Y. enterocolitica is now divided into five biovars 147

CHARACTERS OF GRAM-NEGATIVE BACTERIA

(Nilehn, 1969; Wauters, 1970) by their biochemical characteristics. As the distribution of some of these biovars is epidemiologically quite distinct, we not only mention them but include them also in Table 1.9g. Biovars 1-4 are ornithine-, urease-, cellobioseand sucrose-positive; they give a positive VP reaction at 30 C but are negative at 37 C. Biovars 1—4 can be differentiated by their reactions for lipase, indole, aesculin, salicin and xylose (Table 7.9g). Although Y. enterocolitica has been described as lactose negative, it is not unusual to find biovar 1 strains that are lactose positive; such strains are also likely to ferment raffinose (Cornelis, Luke & Richmond, 1978). Biovar 5 strains (Mollaret & Lucas, 1965), isolated from hares may often give negative reactions for sucrose, ornithine and acetoin (VP) and they always give a negative reaction for trehalose, whereas the other four biovars give positive reactions in these tests. Y. frederiksenii and Y. intermedia can be distinguished from Y. enterocolitica by the production of acid without gas from rhamnose. Y. intermedia, in addition, gives positive reactions with oc-methyl-Dglucoside, raffinose and melibiose. Y. kristensenii and Y. aldovae can be distinguished by their negative reactions with sucrose. They can be differentiated from each other and from sucrose-negative Y. enterocolitica biovar 5 strains by their reactions with cellobiose, rhamnose and the VP test. Y. mollaretii and Y. bercovieri, formerly known respectively as Y. enterocolitica biogroups 3A and 3B, differ from each other in their ability to ferment fucose and sorbose and from Y. enterocolitica sensu stricto in the production of acid from mucate. Y. rohdei differs from Y. enterocolitica sensu stricto in growing in Simmons' citrate medium and in giving negative results in the VP test. Y. pseudotuberculosis strains are urease-positive and motile at room temperature but not at 37 °C; they give negative sucrose, cellobiose and ornithine test reactions. Many of these reactions are temperature dependent and are best carried out at 30 °C. Y. pseudotuberculosis is a homogeneous species with few exceptions. A number of serotypes can be distinguished for epidemiological purposes; strains of serotype III are usually melibiose-negative (Mair, Fox & Thai, 1979). The plague bacillus, Yersinia pestis, which 148

[7.9]

requires haematin (Lapage & Zinnemann, 1971), grows slowly in Hugh & Leifson's OF medium: acid appears first in the open tube and only later in the sealed tube. We interpret this as fermentation following an initial oxidative stage; consequently we describe the organism as fermentative and include it with the Enterobacteriaceae (Tables 1.9a,e). Membership of this family was confirmed by numerical taxonomy (Sneath & Cowan, 1958) and by the antigenic relationships demonstrated by Schiitze (1928, 1932Z?) between Y. pseudotuberculosis and the 'Salmonella' group B, and between Y. pseudotuberculosis and Y. pestis. DNA-DNA relatedness indicates that Y. pseudotuberculosis and Y. pestis constitute a single species and it was proposed that the latter should be regarded as a subspecies of the former (Bercovier et al.9 1980c). However, the proposal has sensibly been rejected because of the medical dangers that might arise through such a change in nomenclature. Mi redefinition: Yersinia. Gram-negative rods; motile or non-motile (temperature dependent). Aerobic and facultatively anaerobic. Catalase-positive; oxidase-negative. Sugars attacked fermentatively, occasionally with production of gas. Grow on MacConkey agar. Type species: Y. pestis; NCTC strain 5923. 7.9.21 Other genera. For the genera of enterobacteria already discussed we have included only those species associated with clinical specimens; however, there are several other species which have not yet been isolated from such material (see Holmes & Gross, 1983; 1990) and these are described in Bergey's Manual (1984). For completeness, we mention briefly other genera, the strains of which are mostly few in number and which have been isolated either occasionally or not at all from clinical material. We do not include them in the Tables but give references for those interested. Budvicia has a single species B. aquatica, which has been isolated so far only from fresh water and small wild mammals. The strains have complex growth factor requirements and are reported to produce H2S, and to hydrolyse urea and O-nitrophenyl-p-D-galactopyranoside. They do not produce acid from glycerol,

ENTEROBACTERIA

maltose, sucrose or trehalose, and do not decarboxylate arginine, lysine or ornithine, or produce phenylalanine deaminase (Bouvet et al., 1985). Ewingella has a single species E. americana, isolated from clinical specimens. The few strains reported are said to be anaerogenic, to produce acid from glucose and to give a positive result in the Voges-Proskaiier test. They do not produce deoxyribonuclease, arginine dihydrolase, lysine decarboyxlase, or ornithine decarboyxlase nor do they ferment arabinose, raffinose, sorbitol or sucrose (Grimont et al, 1983). The reference strains we have examined were indistinguishable from Erwinia herbicola (see Section 7.9.8). Koserella has a single species K. trabulsii, described from 12 strains isolated from clinical material (Hickman-Brenner et al, 1985a). The species, which closely resembles Hafnia alvei, is reported to utilize citrate (Simmons' medium), to give positive results in tests for arginine dihydrolase, lysine decarboxylase, ornithine decarboxylase, fermentation of cellobiose and melibiose and in the methyl red test. The strains give negative results for indole, VP, H2S, urease and phenylalanine deaminase production, and do not ferment glycerol, lactose, sorbitol and sucrose. Leminorella has two species: L. grimontii and L. richardii (Hickman-Brenner et al, 1985b). The first species was described from six strains (four from faeces; two from urine) and the second from four strains from faeces. The former species produces gas from glucose and gives a positive reaction in the methyl red test whereas strains of L. richardii give negative results. Leminorella strains produce H2S and ferment arabinose and xylose but give negative results in most other tests. Moellerella has a single species M. wisconsensis, described from nine strains isolated from faeces (Hickman-Brenner et al, 1984). They are reported to give positive results in Simmons' citrate medium and the methyl red test and to produce acid from lactose and raffinose, but give negative results in most other tests. Colonies on MacConkey Agar are indistinguishable from those of Escherichia coli. We have examined some strains but all of them differed from the original description of the species in fermenting

[7.9] arabinose and not utilizing citrate (Simmons' medium), presumably due to differences in methods. Obesumbacterium has a single 'species', O. proteus, which Priest et al. (1973) divided into groups 1 and 2. Despite their phenotypic similarity and their association with the brewery environment they are phylogenetically different. Indeed, DNA-DNA hybridization reveals that O. proteus biogroup 1 is closely related to Hafnia alvei; Farmer et al. (1985) therefore proposed that it should be classified as a distinct biogroup of H. alvei to be known either as H. alvei biogroup 1 or as the H. alvei-brewery biogroup; O. proteus biogroup 2 would retain the name Obesumbacterium. Strains of both biogroups are difficult to identify as they are slow-growing and fastidious when incubated at 36 °C. Strains of both biogroups ferment D-glucose and D-mannose, but other sugar reactions may vary between the biogroups. Pragia with a single species, P. fontium, has so far been isolated almost exclusively from water in Czechoslovakia. It is said to be motile, able to grow on Simmons' citrate medium and to produce H2S, oxidize gluconate, ferment glucose and galactose and give a positive result in the methyl red test (Aldova et al, 1988). Rahnella has a single species R. aquatilis, which has so far been isolated only from water and soil (Izard et ah, 1979). The reference strains we have examined failed to utilize citrate (Simmons' medium), and did not produce urease, H2S or liquefy gelatin; they were malonate- and ONPG-positive, and fermented several carbohydrates but not adonitol, inositol or starch; gas was not produced from glucose. Xenorhabdus has two species: X. luminescens and X. nematophilus, both so far isolated only from nematodes and the insect larvae they parasitise (Thomas & Poinar, 1979). They are unusual members of the Enterobacteriaceae in giving negative results in most biochemical tests used for differentiation. They do not reduce nitrate or ferment most carbohydrates; acid production from glucose is generally weak or delayed. The former species is bioluminescent and the latter does not produce catalase. 149

CHARACTERS OF GRAM-NEGATIVE BACTERIA

Yokenella has a single species Y. regensburgei, described from eleven strains of which five were isolated from clinical material and six from insects (Kosako, Sakazaki & Yoshizaki, 1984). The strains are reported to resemble H. alvei except for utilizing citrate (Simmons' medium), fermenting cellobiose and melibiose, in giving negative Voges-Proskaiier test results (at 37 °C) and in failing to ferment sorbitol. This organism is probably synonymous with Koserella trabulsii (Hickman-Brenner et ah, 1985a). The characteristics not only of most of the above species, but also of several as yet unnamed groups of enterobacteria and of particular biotypes of some of the better-known species, are given by Farmer et al. (1985). 7.10

A group of difficult organisms {Haemophilus, Gardnerella, Campylobacter, Arcobacter, Helicobacter, Anaerobiospirillum, Eikenella, Streptobacillus, Legionella, Mycoplasma) Characters common to members of the group: Gram-negative rods. Exacting though differing requirements for isolation and continued growth in subculture. These organisms have little in common except for the demands they make on the skill and ingenuity of bacteriologists. If, as in the first edition of this Manual, we had a chapter on miscellaneous bacteria and those of uncertain taxonomic position all these would have qualified for it, but as we are more concerned with identification than with taxonomic position, we have collected them into a 'group of difficult organisms'. They are difficult in many ways, not least in being fastidious in their nutritional and atmospheric requirements. Neither Eikenella nor Campylobacter can be isolated from material incubated aerobically, and anaerobic culture is not much more successful. Eikenella grows in air with added CO2 while Campylobacter prefers air with only 5-10% oxygen in it, as also do Arcobacter and Anaerobiosprillum though the latter requires anaerobic conditions for subculture. Haemophilus species are not so fussy about their atmospheric surroundings but they are demanding in their X- and V-factor requirements. Streptobacillus prefers anaerobic conditions and 150

[7.9]

requires a medium much richer in protein than is usually provided; like other members of the group its growth is improved by the addition of carbon dioxide. Legionella has an absolute nutritional requirement for L-cysteine and iron for primary isolation and often for subculture. And Mycoplasma is in a class of its own and requires special methods for characterization. 7.10.1 Haemophilus (Tables 7.10a,*} is still defined as Gram-negative coccobacilli or rods that require one or both of the two accessory growth factors X (haemin or other porphyrins) and V (Coenzyme 1: nicotinamide adenine dinucleotide (NAD) or its phosphate). These species will not grow on the media used in conventional biochemical tests, and X and V factor supplements are needed for the indole and carbohydrate tests shown in Table 1.10b. The Xfactor requirement reflects their inability to synthesise the porphyrins necessary for their respiratory enzymes: it is usually demonstrated by the absence of growth on porphyrin-deficient but otherwise nutritionally adequate media except near paper discs or strips impregnated with X- and, if necessary, V-factor. However, most basic liquid and solid media contain traces of X-factor, as do some brands and batches of peptone. Zinnemann (1960) recommended Yeastrel (Yeast Extract) Agar as a consistently X-free medium for testing this requirement of Haemophilus species. Doern and Chapin (1984) found Trypticase Soy Agar to be more reliable than Brain Heart Infusion or Mueller-Hinton agar for this purpose. Kilian (1974) doubted whether any complex medium which otherwise satisfied the growth requirements of haemophili was completely free of X-factor. He claimed that more reliable results for X-factor requirements were obtained by testing the ability of haemophili to synthesize porphyrins from 8-amino laevulinic acid (ALA) and this was confirmed by Tebbutt (1983). A disc method for the ALA porphyrin test (Lund & Blazevic, 1977) and a porphyrin test medium (Gadberry & Amos, 1986) have also been described. V-factor acts as a coenzyme or catalyst in oxidation-reduction reactions and this requirement of strains can be determined either by means of impregnated paper discs or strips, or less specifically by demonstrating satellitism near the growth of Staphylococcus aureus which produces V-factor in

[7.10]

A GROUP OF DIFFICULT ORGANISMS

Table 7.10a. Second-stage table for Haemophilus, Eikenella, Gardnerella, Campylobacter, Helicobacter, Streptobacillus, Arcobacter, Anaerobiospirillum, Legionella and Mycoplasma

Motility Growth in air Growth under anaerobic conditions Catalase Oxidase Swelling of microbial cell Colony adherent to medium Growth at 42 °C Growth favoured by O2 Growth favoured by CO2 Growth improved by blood/serum Growth improved by moisture (closed jar) Carbohydrates [VIOI-] Glucose, acid from Starch hydrolysis Nitrate reduced Indole Gelatin liquefied Urease H2S Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Zone of inhibition with metronidazole (50 jig) 1 2 3 4 5 6 7 8 9

1

2

3

4

5

6

_ + + D D 7 D

_

_ + + _ w 7 + + F + + _ 7 -

+ D D +/ D +8 + + + D _ D D 7 7 7 d

_ + + _ + + + + +a V + + _ + 7 7 7

_ + + _ + 7 7 7 + 7 VI+ +c 7 7 D 7 D 7 7 7

+ F + 7 + D D D D D 7

+ + + w 7 + + + _ + _ (w)/+ d -

+

7

8

9

+ + d d + w/-

+

+ + 7 _ + _ D 7 7 7

+ + _ _ + 7 + 7 F + _ 7 7 7 7

+

+

+ d d + 7 7 + _ _ + NT -

Haemophilus spp. Eikenella corrodens; HB-1 Gardnerella vaginalis; 'Corynebacteriwn vaginale' Campylobacter and Helicobacter spp.; V. fetus Streptobacillus moniliformis; 'Actinobacillus muris'; 'Actinomyces muris' Mycoplasma spp.; Acholeplasma spp Arcobacter spp. Anaerobiospirillum succiniciproducens Legionella spp.

a Requirement for growth b Blood requirement is for haematin (X factor) c False positive reactions can occur due to amylases in serum d Haemin usually required for growth under aerobic conditions e Cysteine and iron salts required for growth / C. curvus and C. rectus positive in modified test of Tarrand & Groschel (1982) g O2 essential for growth but >10% is toxic h Optimal O2: 3-10% NT not testable by usual method Other symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

excess of its own need. Since V-factor is susceptible to heat, media in which all the constituents have been autoclaved will be reliably free from it. However, consistent results in tests for these growth factor requirements depend on the use of small inocula to avoid any carry-over. Occasional strains in other unrelated taxa sometimes exhibit growth-factor

requirements similar to those of Haemophilus species; for example, occasional strains of Eikenella corrodens have an apparent requirement for X-factor. This can cause confusion and illustrates the danger of basing identification of the genus Haemophilus solely on X- and V-factor requirements. Haemophilus species are fastidious not only in 151

CHARACTERS OF GRAM-NEGATIVE BACTERIA

their X- and V- requirements but also in their need for other nutritional factors, which vary among the species. H. ducreyi and H. parasuis have a requirement for serum; so does H. paragallinarum, which also has a requirement for NaCl (Rimler et al., 1977) though it is not halophilic in the usual meaning of the term. Raised carbon dioxide tension improves growth, and may be necessary for a number of species, including H. aphrophilus, H. paraphrophilus, H. paraphrohaemolyticus and H. paragallinarum. This apparent need for CO2 may be fulfilled by incubating cultures in the moist atmosphere of a sealed jar. However under the usual conditions of aerobic incubation recently isolated strains may grow only feebly or not at all; added CO2 should therefore be used for primary isolation and it may prove beneficial for subsequent subcultures of haemophili (Frazer, Zinnemann & Boyce, 1969). The optimum temperature for growth is 35-37 °C. For culture, ordinary Blood Agar medium is not suitable unless heated to form 'Chocolate Agar'; this not only releases the V-factor from disrupted erythrocytes but also inactivates any enzymes which might affect X-factor. For primary isolation of haemophili from the respiratory tract, selective media containing for example crystal violet (Turk, 1963), bacitracin (Baber, 1969) and other antibacterial agents (Chapin & Doern, 1983) can be used to inhibit overgrowth by other members of the normal flora. Some species of Haemophilus, notably H. influenzae as well as H. parasuis, H. gallinarum and H. avium, can produce polysaccharide capsules which are antigenically type-specific and related to pathogenicity. Occasional capsulated strains of H. parainfluenzae have also been described (Sims, 1970). Such strains exhibit iridescence on transparent media such as that of Levinthal (1918) or the peptic digest of Fildes (1920). With H. influenzae, six capsular types (a-f) can be identified (Pittman, 1931) of which type b is the most frequent among capsulated strains isolated from infections in man. Haemophilus species can be differentiated by their growth-factor requirements and cultural characteristics (Table 7.10Z?). The species commonly encountered in medical laboratories can usually be identified satisfactorily by these methods alone. With a difficult strain, or in order to confirm the identity of an unusual species, further biochemical tests may be neces152

[7.10]

sary; these include carbohydrate utilization tests in Phenol Red Broth sugars supplemented with 10 jig/ml each of X- and V-factors (Kilian, 1976). The results can usually be read after 24 hours incubation, though some species, notably H. segnis, give weak reactions. As carbohydrate utilization and concomitant acid production by haemophili are, to some extent, dependent on the medium used, the results of these tests should be interpreted with caution. Most strains of H. parainfluenzae, H. haemolyticus, H. aphrophilus and H. paraphrophilus produce small amounts of gas (H2 and CO2) from glucose (Kilian, 1976); other species of Haemophilus do not produce any gas. As facultative anaerobes, all Haemophilus species can ferment glucose anaerobically (Sneath & Johnson, 1973) but the exact mode of carbohydrate breakdown is not known so that the description 'fermentation' may simply mean acid production by unknown metabolic means. The catalase activity of haemophili (Table 7.10fr) may vary with the cultural conditions; for example, some apparently catalasenegative strains may give a positive reaction if provided with sufficient haemin (Biberstein & Gills, 1961). The oxidase reaction is always positive with H. influenzae and negative with H. aphrophilus', the other species do not give consistent results. Rapid test kits for identification of Haemophilus species have also been described (Janda, Malloy & Schreckenberger, 1987; Murphy etal, 1990). Three biochemical reactions - indole production and urease and ornithine decarboxylase activity form the basis of a scheme devised by Kilian (1976) for biotyping H. influenzae and H. parainfluenzae. These tests, which depend on the presence of preformed enzymes, are performed in media heavily inoculated with bacterial growth from an 18 hour agar culture. The results can be read after 5 hours incubation, though the ornithine decarboxylase test may give a clear reaction only after overnight incubation. With H. influenzae, Kilian distinguished five biotypes (I-V) which showed some correlation with different infections. Three further biotypes of H. influenzae were described subsequently by Oberhofer & Back (1979), Gratten (1983) and Sottnek & Albritton (1984). The majority of clinical isolates of H. influenzae belong to biotypes I, II and III; most isolates from low-grade and chronic infections are non-capsulated strains of biotypes II and III whereas

A GROUP OF DIFFICULT ORGANISMS

those from acute infections such as meningitis are usually caused by capsulated strains of biotype I. Other studies purport to show a correlation between biotype, serotype, antimicrobial susceptibility (Granato, Jurek & Weiner, 1983) and the source of the isolates (Albritton et al, 1978). With H. parainfluenzae, eight biotypes have been described (Kilian, 1976; Oberhofer & Back, 1979; Sturm, 1986). It is not clear, however, if biotyping has contributed greatly to our understanding of Haemophilus infections. For epidemiological studies, subtyping schemes based on outer membrane proteins (Loeb & Smith, 1980), on lipopolysaccharides (Inzana, 1983) or on isoenzyme profiles (Musser et al, 1985) are more sensitive than biotyping. Metabolic adaptations among Haemophilus species can occur in vitro which, by reducing or abolishing specific requirements, may be responsible for some of the conflicting statements in the literature. H. aphrophilus was originally isolated from cases of infective endocarditis by Khairat (1940) and was so named because it needed X-factor and CO2 for growth, though the X-factor requirement may be lost on subculture. King &Tatum (1962) were unable to confirm the X-factor requirement of strains sent to them, but they pointed out that these strains had been subcultured several times and so may well have lost it. They thought that the strains had much in common with those of Actinobacillus actinomycetemcomitans, but had relatively few of the recognized characters of either Haemophilus or Actinobacillus. White & Garrick (1963) showed that H. aphrophilus possessed all the enzymes of the haemin-biosynthetic pathway characteristic of species not requiring X-factor; indeed, it gave a positive but weak reaction in the porphyrin ALA test. The reason for the apparent Xfactor requirement on primary isolation is unknown. Boyce, Frazer & Zinnemann (1969) discussed the difficulties of classifying strains that adapt quickly to in vitro conditions and concluded that because the majority of bacterial cells in each culture were incapable of synthesising X-factor, H. aphrophilus should be regarded as essentially X-dependent. Since we try to show the characteristics of freshly isolated strains, this X-dependence is recorded in Table 7.10£. DNA-DNA hybridization studies indicate that H. aphrophilus and H. paraphrophilus are closely related (Potts et al, 1986; T0njum, Bukholm & B0vre,

[7.10] 1990), thus adding weight to the suggestion of Potts & Berry (1983) that one of them could be regarded as a subspecies of the other. However, as H. aphrophilus is X-factor and H. paraphrophilus V-factor dependent, we continue to regard them as separate species. There has been much debate as to whether H. aegyptius (the Koch-Weeks bacillus) should be regarded as a species separate from H. influenzae. The dilemma is not surprising since transformation studies (Leidy, Hahn & Alexander, 1959; Leidy, Jaffee & Alexander, 1965) and DNA hybridization (Casin, Grimont & Grimont, 1986) have shown a high degree of relatedness. Pittman and Davis (1950) differentiated H. aegyptius from H. influenzae on the basis of acid production from xylose and agglutination of human red cells. Kilian (1976) considered H. aegyptius to be synonymous with H. influenzae biotype III. In a further taxonomic study of 112 strains of Haemophilus isolated from conjunctivae, Kilian et al. (1976) again stated that H. aegyptius should be regarded as a haemagglutinating variety of H. influenzae biotype III. However Mazloum et al. (1982), in a study of 29 isolates from cases of acute conjunctivitis in Egypt, claimed that strains of H. aegyptius could be distinguished from those of H. influenzae biotype III by their inability to ferment xylose or to grow in Trypticase Soy Agar despite added X- and V-factors, and by their susceptibility to troleandomycin. Kilian & Biberstein (1984) stated that only sensitivity to troleandomycin would unequivocally distinguish between them but Carlone et al. (1985) regarded this test as unreliable; their studies did however reveal distinct differences in the protein profiles of the outer membranes of the two organisms despite their close genetic relatedness. Brenner et al. (1988) accordingly adopted the term 'H. influenzae biogroup aegyptius' for strains which would formerly have been called H. aegyptius. However, the view of Casin, Grimont & Grimont, (1986) that the differences in pathogenicity between the two organisms are of little relevance in classification accords with the probability that they represent one taxon; for this reason, we do not include H. aegyptius in Table 7.10b. This organism has nevertheless been associated with the infection described recently as 'Brazilian purpuric fever' (Brenner et al, 1988). Haemophili isolated from swine and fowl have 153

[7.10]

CHARACTERS OF GRAM-NEGATIVE BACTERIA

Table 7.10b. Third-stage table for Haemophilus, Gardnerella, Actinobacillus actinomycetemcomitans and Eikenella corrodens l V-factor requirement X-factor requirement ALA —> porphyrins Catalase Oxidase Growth on Chocolate Agar Haemolysis CO2 improves growth Indole Urease Ornithine decarboxylase *Carbohydrates, acid from glucose fructose galactose inulin lactose mannitol raffinose ribose sorbitol sucrose trehalose xylose

+ + + + + Da Da Da

+ + + + + +b d + -

+ + + + + _ -

+ _ + dc d d _ -

+ +

+ w +

_

_ _ + d

+ +

(w/-) _ _

+ +

1 Haemophilus influenzae 2 Haemophilus haemolyticus 3 Haemophilus haemoglobinophilus; i H. canisy 4 Haemophilus ducreyi 5 Haemophilus aphrophilus 6 Haemophilus parainfluenzae

d + +

7 8 9 10 11 12 13

w _ _ +e + _ + + + + _ + + + + -

+ + d + + Da Da

+

+

10

11

12

13

+

+

+

+

14*

15

16

w/+e

+e

Da

+ + + + -

Haemophilus Haemophilus Haemophilus Haemophilus Haemophilus Haemophilus Haemophilus

w w w

parahaemolyticus; paraphrohaemolyticus paraphrophilus segnis parasuis paragallinarum avium

14 Gardnerella vaginalis*; 'Corynebacterium vaginale' 15 Actinobacillus actinomycetemcomitans 16 Eikenella corrodens

* Note. Phenol red broth sugars supplemented with X and V factors are essential for Haemophilus spp. Carbohydrate reactions are not reliable for Gardnerella. a Reactions vary with biotypes b Haemolysis with bovine or sheep blood; variable with horse blood. May lose haemolytic activity on subculture c Optimum temperature for growth 33 °C, but very slow. Growth improved with addition of 1% Iso Vitalex (Hammond et al., 1978) d Haemin usually required for growth under aerobic conditions e Positive in CO2 / Serum improves growth g p-haemolysis with human and rabbit blood; haemolysis variable with horse blood h Requires haemin-containing media on primary isolation / Slight greening of medium may occur around colonies Other symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

also posed considerable problems of classification and nomenclature, which were discussed at some length by Biberstein & White (1969). 'Haemophilus influenzae suis} was proposed by Lewis & Shope (1931) for an organism isolated from swine influenza in America. This organism, usually referred to as H. 154

suis, required both X- and V-factors and was virtually indistinguishable from indole-negative strains of H. influenzae. However, the original cultures of Lewis & Shope when re-examined by Matthews & Pattison (1961) were found to be no longer X-dependent, possibly because of adaptation in vitro. Biberstein &

A GROUP OF DIFFICULT ORGANISMS

White (1969) proposed a new species, Haemophilus parasuis, for strains that did not require haemin but which were otherwise identical to H. suis. Since then it has become clear that the overwhelming majority of strains isolated from swine require only V-factor and thus, according to the usual convention for naming Haemophilus species, should be called H. parasuis. Moreover most of the strains in culture collections labelled H. suis do not need X-factor and are therefore H. parasuis (Kilian & Biberstein, 1984). For these reasons we include only H. parasuis in Table 7.10fr. The organism formerly classified as Haemophilus pleuropneumoniae has been transferred recently to the genus Actinobacillus (Pohl et al, 1983) on the basis of its phenotypic characters and DNA relatedness to Actinobacillus lignieresii; however the emended species A. pleuropneumoniae includes both V-factor requiring and V-factor independent biovars. Haemophili isolated from fowl pose problems similar to those from swine. There are conflicting reports on the growth-factor requirements of H. gallinarum, the fowl coryza bacillus. De Blieck (1932) stated that it required both X- and V-factors and this was confirmed by Schalm & Beach (1936) and by Delaplane, Erwin & Stuart, (1938) who showed also that H gallinarum had an obligate requirement for sodium chloride (0.5-2.5%). However later studies have failed to demonstrate any strains of avian haemophili which required X-factor for growth (Page, 1962; Roberts, Hanson & Timms, 1964). Biberstein & White (1969) accordingly proposed the name Haemophilus paragallinarum for X-independent haemophili which were otherwise identical to H. gallinarum. Piechulla, Hinz & Mannheim (1985) suggested on the basis of DNA homology that H. paragallinarum is more closely related to the genus Actinobacillus than to Haemophilus. Page (1962) divided the avian haemophili into two groups. The first group, H. paragallinarum, was catalase-negative, grew in air with 5% CO2 and caused infectious coryza; the second group of strains, which were catalase-positive, grew in air and were non-pathogenic, was subsequently allocated to a new species, H. avium, by Hinz & Kunjara (1977). DNA-DNA hybridization (Mutters et al., 1977) indicated that H. avium contained three clusters, which were genetically closer to Pasteurella multocida than to Haemophilus influenzae. Mutters et

[7.10] al. (1977) accordingly suggested reclassifying H. avium into three new taxa, Pasteurella avium, P. volantium and Tasteurella species A'. However, on the basis of their phenotypic characters not all strains of H. avium could be assigned to Pasteurella in this way (Blackall, 1988). It is thus clear that further work on haemophili from swine and fowl is needed to clarify the present situation. Casin et al. (1985) suggested that H. ducreyi, which is phenotypically distinct from other Haemophilus species, is misplaced in that genus because of the low level of DNA homology. This is supported by Carlone et al. (1988) who found that the respiratory quinone content of H. ducreyi differed significantly from that of other haemophili. Indeed Hollander, Hess-Reihse & Mannheim (1981) suggested that Haemophilus species should be reclassified on the basis of their respiratory quinones. The long-standing debate on the correct classification of the organism previously labelled 'Haemophilus vaginalis' and subsequently as 1 ' Corynebacterium vaginale' has been resolved at last by the creation of a new genus with a single species, Gardnerella vaginalis (Greenwood & Pickett, 1980; Piot et al., 1980). We describe this Gram-variable rod in Section 7.10.2 but, for convenience, we also record its characteristics in Tables 7.10a, b. Other species have been described, but by present standards they do not warrant inclusion in the genus Haemophilus as none of them requires X- or V-factors for growth. They include 'Haemophilus somnus' (Bailie, Coles & Weide, 1973), 'Haemophilus agnV (Kennedy et al., 1958) and 'Haemophilus equigenitalis' (Taylor et al., 1978). An organism requiring Vfactor isolated from the small intestine of rabbits with mucoid enteritis was named Haemophilus paracuniculus by Targowski & Targowski (1979), but its pathogenic role and correct classification have not yet been confirmed. Potts, Zambon & Genco, (1985) have proposed that Actinobacillus actinomycetemcomitans should be reassigned to the genus Haemophilus as H. actinomycetemcomitans. DNA-DNA hybridization studies of A. actinomycetemcomitans, H. segnis, H. aphrophilus and H. paraphrophilus showed considerable homology consistent with placing them in a single genus. Antigens common to all these species were also found by immunodiffusion. However, 'H. acti155

CHARACTERS OF GRAM-NEGATIVE BACTERIA

nomycetemcomitans" does not require either X- or Vfactors and it thus does not easily fit into the genus Haemophilus as currently defined, but for comparison we include it in Table 1.10b. Minidefinition: Haemophilus. Gram-negative rods or coccobacilli; often markedly pleomorphic; non-motile. Aerobic and facultatively anaerobic. Oxidase and catalase reactions vary between species and strains. Nitrate reduced to nitrite: Fastidious; require media containing X- and V-factors and undefined constituents of blood and serum. Attack sugars fermentatively. Type species: H. influenzae; NCTC strain 8143. 7.10.2 Gardnerella (Table 7.10a) has caused much controversy since it was first described by Leopold in 1953. Gardner & Dukes (1954) and Lutz, Grooten & Wurch, (1956) noted that a similar organism was frequently associated with non-specific vaginitis or anaerobic vaginosis; Gardner & Dukes (1955) regarded it as the causal organism and, on the basis of its microscopical appearance and apparent growth requirement for blood, proposed the name Haemophilus vaginalis. However, neither X-(haemin) nor V-(nicotinamide adeninine dinucleotide) factor was required for its growth (Edmunds, 1962; Dunkelberg & McVeigh, 1969); one or other of these is essential for inclusion in the genus Haemophilus as currently defined. It is, however, fastidious in its growth requirements, needing thiamine or related substances as well as purines and pyrimidines (Dunkelberg & McVeigh, 1969). Lapage (1961) confirmed that it did not require X- or V-factors and suggested it might be a Corynebacterium', this was supported on Gram-staining and morphological grounds by Zinnemann & Turner (1963) who recommended its reclassification as Corynebacterium vaginale. Although Dunkelberg & McVeigh (1969) agreed with this proposal it failed to gain universal approval. 'C. vaginale' is catalase-negative, whereas most corynebacteria are positive and the cell wall of ' C vaginale', unlike that of corynebacteria, lacks teichoic acid and arabinose (Keddie & Cure, 1978). Not surprisingly, the difficulty receded when Greenwood & Pickett (1979), proposed a new genus, Gardnerella, for it based on cell wall composition, DNA homology and electron microscopy. 156

[7.10]

Subsequent taxonomic studies (Greenwood & Pickett, 1980; Piot et al., 1980) confirmed that Gardnerella vaginalis is not related to the genus Haemophilus or to Corynebacterium, and the name G. vaginalis has since been widely accepted. The precise taxonomic position of G. vaginalis remains uncertain; indeed, whether it is Gram-positive or Gram-negative is still unresolved and it is included among both the Gram-positive and the Gram-negative organisms in Bergey's Manual of Systematic Bacteriology by Greenwood & Pickett (1984, 1986). Based on the amino acid composition of the cell wall Criswell et al. (1971) stated that G. vaginalis was Gram-negative but their results were criticised by Easmon & Ison (1984) as they had also failed to detect the diaminopimelic acid known to be present in the cell walls of Escherichia coli and Bacillus megaterium. Conversely, Harper & Davis (1982) considered the amino acid composition of the cell wall to be that of a Gram-positive organism. Electron microscopy has failed to resolve this dispute, though Reyn, Birch-Anderson & Lapage, (1966) reported that the structure of the cell wall and septa was more like that of Gram-positive organisms; however, Criswell and her colleagues (1971, 1972) observed an unusual trilaminar cell-wall appearance more closely resembling that of Gram-negative bacteria. Greenwood & Pickett (1980) concluded that the cell wall was more like that of Gram-negative than of Gram-positive organisms, but it was not 'typical'. The status of the Gram-reaction has yet to be resolved but in practice, Gram-stained smears of fresh cultures show mostly Gram-positive bacteria and ageing cultures significantly more Gram-negative organisms; in smears from clinical material both forms occur. We have placed it among the Gram-negative organisms in Table 7.10a though we refer to it in Section 6.5 and include it also in Table 6.5b. G. vaginalis is aerobic and facultatively anaerobic though obligate anaerobic strains have been reported (Malone et al., 1975); growth is enhanced by the addition of 5-10% CO2. It produces diffuse p haemolysis on human or rabbit Blood Agar but not on sheep Blood Agar; haemolysis on horse Blood Agar is variable (Greenwood et al., 1977). This character is exploited for selective isolation of the organism from clinical specimens; and the use of layered human Blood Agar plates, with the addition of Tween

A GROUP OF DIFFICULT ORGANISMS

80, has now largely superseded the Peptone Starch Glucose Agar medium described by Dunkelberg & McVeigh (1969) and Totten et al. (1982). Smith (1979) and Piot et al. (1980) noted, however, that occasional strains of lactobacilli and streptococci from vaginal specimens also produced similar phaemolysis. Definitive identification of G. vaginalis is difficult as there are no wholly reliable test characters. It ferments carbohydrates without gas production, the major end-product being acetic acid (Moss & Dunkelberg, 1969) but the reported reactions for some carbohydrates vary (Edmunds, 1962; Greenwood & Pickett, 1979; Piot et aL, 1980) and are not always reproducible (Taylor & Phillips, 1983) probably because of differences in the test methods and media used. Furthermore, the carbohydrate reactions do not reliably differentiate G. vaginalis from lactobacilli among the vaginal flora. We do not therefore recommend their use in routine diagnostic laboratories except for non-genital isolates (Piot, 1985). Yong & Thompson (1982) described a rapid (one hour) micromethod for hippurate hydrolysis and the fermentation of starch and raffinose; the medium described by Dunkelberg, Skaggs & Kellog (1970) modified by Greenwood & Pickett (1979) can also be used for these tests. Heavy inocula may help to minimize the variability of the biochemical results (Piot & vanDyck, 1981). As simple test characters, P-haemolysis on human, but not on horse Blood Agar, the Gram-reaction, the microscopical appearance, colonial morphology, and negative oxidase and catalase reactions have all been recommended for reliably identifying G. vaginalis (Ison et aL, 1982; Shaw et aL, 1981; Piot et aL, 1982). Greenwood and Pickett (1979) used hippurate hydrolysis and P-haemolysis to separate G. vaginalis from other catalase- and oxidase-negative organisms; and Jolly (1983) considered them indispensable for the accurate identification of G. vaginalis in the routine laboratory. Taylor and Phillips (1983) however found that hippurate hydrolysis did not reliably distinguish between G. vaginalis and catalase-negative coryneform organisms. The enzymatic O-nitrophenol tests for the detection of a- and (3-glucosidase were also recommended by Piot et aL (1982) but Taylor & Phillips (1983) found these unhelpful. G. vaginalis is susceptible to metronidazole and

[7.10] trimethoprim but resistant to sulphonamides (Bailey, Voss & Smith, 1979; Piot et aL, 1980); despite our reservations about such tests, they are helpful in preliminary identification. In addition, Taylor & Phillips (1983) claim that p-haemolysis on human Blood Agar medium, the failure to grow on Nutrient Agar or in the presence of 2% (w/v) NaCl, and the inability to produce lactic acid from glucose will differentiate G. vaginalis from lactobacilli and catalase-negative coryneform organisms isolated from vaginal secretions. In routine laboratories, p-haemolysis on human but not horse Blood Agar, the Gram-stain reaction and morphology as well as susceptibility of most strains to discs of metronidazole (50 jig) and trimethoprim (5 |ig) should identify satisfactorily more than 90% of isolates. Biotyping schemes for G. vaginalis have been described by Piot et aL (1984) and by Benito et aL (1986). Minidefinition: Gardnerella. Gram-negative or Gram-variable rods or coccobacilli; often markedly pleomorphic; non-motile. Aerobic and facultatively anaerobic. Oxidase- and catalase-negative. Fastidious growth requirements. Attacks sugars fermentatively. Gas is not produced. Type species: G. vaginalis; NCTC strain 10287. 7.10.3 Eikenella (Table 7.10a) is the name now given to a group of facultatively anaerobic Gramnegative non-sporing rods that will grow aerobically from the first subculture, though anaerobic conditions may be necessary for isolation (Jackson & Goodman, 1972); these organisms form the HB-1 strains of King & Tatum (1962). The genus was named after Eiken who, in 1958, gave the specific epithet to the organism (which he had placed in Bacteroides). According to Jackson & Goodman two rather different organisms, both of which produced 'pitting' on the surface of the medium during growth, have been named Bacteroides corrodens', one is a strict anaerobe (and remains so) which is urease-positive; the other {Eikenella corrodens) is facultatively anaerobic and urease-negative. A few points of difference between these organisms are shown in Table 1.2a. Hill, Shell & Lapage, (1970) did not find any of the strictly anaerobic organisms among the NCTC strains, which had been isolated mostly in the U.K. In describing these organisms Jackson & Goodman 157

[7.10]

CHARACTERS OF GRAM-NEGATIVE BACTERIA

Table 7.10c. Third-stage table for Campylobacter, Helicobacter, Arcobacter and Anaerobiospirillum 1 2 Rapid coccal transformation Spreading surface growth Growth at 25 °C Growth at 43 °C Catalase Growth inhibited by: nalidixic acid6" cephalothin^ Hippurate hydrolysis^ Indoxyl acetate hydrolysis* Nitrate reduced'? H2S from FBP^ H2S from TSI Growth in the presence of: triphenyltetrazolium chloride 0.04% (agar) 1 % glycine 1.5% sodium chloride (agar) Growth under anaerobic conditions Urease' 1 2 3 4 5 6 7

Campylobacter fetus subsp. fetus Campylobacter fetus subsp. venerealis Campylobacter hyointestinalis Campylobacter lari; C. laridis Campylobacter coli Campylobacter jejuni subsp. jejuni Campylobacter jejuni subsp. doylei

* a b c d e /

Rapid disc method of Mills & Gherna (1987) Scraped colonies appear yellow Smells of hypochlorite Growth also at 15 °C Requires H2 or formate 30 jig disc, any zone of inhibition Method of Hwang & Ederer (1975)

3

4

5

6

7

10

- - + W+ + W - - - W + + W + + + d f l - - - - — — w + + + — d + + + + + + d w/-

_ + d +j _

_ -

+d -

d

d +

+ +

+k _

+^ —fn

d +J _

+i

di

d/

+ +

+ + -

— _

8 Campylobacter upsaliensis 9 Campylobacter sputorum subsp. sputorum 10 Campylobacter concisus 11 Helicobacter pylori 12 Helicobacter cinaedi; Campylobacter cinaedi g h / j k / m

— _

_ + —

_ d d - -

11

12

13

14

15

w

w

-

+c d

w d d d -

d

-

-

+

d

+

-

d

+

-

-

-

?

+ + -

+ + -

d d d

? ? ?

4^

_

_

13 Helicobacter fennelliae; Campylobacter fennelliae 14 Arcobacter cryaerophilus; Campylobacter cryaerophila 15 Anaerobiospirillum succiniciproducens

Method 5-Cook (1950); C3.39 Method 7 - L i o r (1984); C3.3O Blackening where there is water of synaeresis Requires fumarate (0.2%) or nitrate (0.1 %) Positive in the presence of trimethylamine N-oxide (0.1%) Christensen's urea medium without agar (A2.6.41) A urease-positive subgroup has been described (Bolton et ai, 1985)

Note. Use Mueller-Hinton or Blood Agar for these tests unless otherwise stated. Raising the agar concentration to 2% facilitates reading zones of inhibition by preventing growth spreading. Organisms 7, 8 and 11-15 require blood for reliable growth and plates should be moist. Other symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

(1972) and Prefontaine & Jackson (1972) distinguished the facultative anaerobe by its DNA G+C content of 28-30% compared with 57-58% for the strictly anaerobic B. corrodens; the fatty acid composition of the organisms as determined by gas chromatography was also different. These are esoteric differences and at present still only of theoretical importance, but they do indicate that there are two colonially similar but otherwise different organisms. Eikenella corrodens has been isolated alone or in mixed culture from pus from various abscesses, pleu158

ral fluids, sputum, tonsils, the ear and the nose; Jackson & Goodman (1972) regard it as an opportunist pathogen but Riley, Tatum & Weaver (1973), with information about 500 strains available to them, thought that it was a normal inhabitant of the pharynx and alimentary canal. For culture, Blood Agar incubated in an atmosphere enriched with 5-10% CO2 in a closed jar is satisfactory. Jackson & Goodman found growth was improved by 0.1% KNO3, and better with 1% oxygen than under strictly anaerobic conditions. Although

A GROUP OF DIFFICULT ORGANISMS

anaerobic methods using a closed jar seemed to be essential for isolation, it was not clear whether the anaerobiosis, the moisture, the CO2, or a combination of these, were the essential factor(s). Haematin seems to be essential for the growth of subcultures in air. The organism was so named because of the adherent nature of the wrinkled colonies which 'dig' into (corrode) the surface of agar media. Some colonies are not wrinkled, and these are not adherent; they breed true and have the same biochemical characters as the wrinkled colony form. Biochemically, E. corrodens is rather inactive and for that reason has been likened to brucellas and even placed in the same family. Like Campylobacter (Table 7.10c) Eikenella grows better in semisolid than in any liquid medium. Minidefinition: Eikenella. Gram-negative rods; non-motile. Facultatively anaerobic on first isolation; soon become aerobic. Catalase-negative; oxidase-positive. Do not attack carbohydrates. Type species: E. corrodens; NCTC strain 10596. 7.10.4 Campylobacter (Tables 7.10a, c). Since publication of the second edition of this Manual, knowledge of this genus has advanced considerably and Campylobacter jejuni is now recognized as a common cause of acute enterocolitis in man. Several new species have been described and the status of earlier named species has been clarified. They formerly occupied a place within the genus Vibrio as the so-called microaerophilic members exemplified by the animal pathogen 'Vibrio fetus', but there is every justification for their inclusion in the genus Campylobacter as proposed by Sebald & Veron (1963). In general, campylobacters do not grow in conventional aerobic or anaerobic culture systems, a fact that undoubtedly delayed their recognition as important human pathogens. They grow best in 5-10% oxygen, though some species, notably C sputorum (Loesche, Gibbons & Socransky, 1965), grow anaerobically in the presence of fumarate or nitrate. Most strains grow satisfactorily in sloppy media (0.16% agar) suitably supplemented and incubated aerobically. The intolerance of most species to atmospheric oxygen is due to their extreme sensitivity to superoxides and free radicals despite their possession of catalase and superoxide dismutase (Hoffman et al., \919a,b)\ culture

[7.10] media must therefore contain agents suitable for neutralizing such toxic substances (George et al. 1978). Whole blood is perhaps the most effective agent, but haemin, inorganic iron salts, pyruvate and charcoal among others have a similar effect. A striking character of campylobacters is their helical or curved shape. Long spiral forms can resemble spirochaetes superficially, but campylobacters have flagella, usually single, at one or both poles (monotrichate or amphitrichate) and are highly motile, spinning around their long axes and frequently reversing direction. The type species, C. fetus, of which there are two subspecies, causes infectious abortion of cattle and sheep, but it rarely infects man. C. fetus subsp. fetus occasionally causes systemic infection in immunodeficient patients. C. hyointestinalis (Gebhart et al., 1985) is closely related to C. fetus. Although more usually encountered in the intestinal tracts of pigs and cattle, it is occasionally isolated from patients with diarrhoea (Edmonds etal, 1987). C. jejuni and C. coli - collectively known in obsolete nomenclature as 'C fetus subsp. jejuni' - are the species most often encountered in medical laboratories as a cause of acute enterocolitis. They are distinguished from C. fetus and most other campylobacters by their high optimum growth temperature (42 °C), a feature described by King (1962) who called them 'related vibrios'. C. jejuni has two subspecies: subsp. jejuni - the familiar cause of enterocolitis in man; and subsp. doylei (Steele & Owen, 1988) - a more fastidious and slower growing organism which does not grow at 43 C or reduce nitrate. C. lari (previously termed 'C. laridis' by Benjamin et al., 1983) is also 'thermophilic' like C. jejuni subsp. jejuni and C. coli but is of low virulence and encountered only occasionally in man. Several biotyping schemes have been described for these 'thermophilic' campylobacters. We mention two: that of Lior (1984), incorporating the modified DNase test of Lior & Patel (1987), which divides C. jejuni into four biotypes (I-IV) and C. coli and C. lari each into two biotypes; and the more complex but more discriminatory combined identification and 'resistogram' typing scheme of Bolton, Holt & Hutchinson (1984). Although serotyping is usually preferred for the differentiation of campylobacter strains, biotyping is a useful alternative or adjunct to it. C. upsaliensis, the name for 159

CHARACTERS OF GRAM-NEGATIVE BACTERIA

the 'CNW' (catalase-negative/weak) group of Sandstedt, Ursing & Walder, (1983) is related to the 'thermophilic' campylobacters, though not all strains grow at 43 °C. Originally isolated from dogs, C. upsaliensis appears to be enteropathogenic for man (Patton et al, 1989; Goossens et al, 1989, 1990). As primary isolation usually requires the use of selective filtration and non-selective media incubated at 37 °C (Bolton et al, 1988; Goossens et al, 1990), it is seldom detected by conventional methods used for C. jejuni and C. coli. Like C. jejuni subsp. doylei it is slower growing and more fastidious than C. jejuni or C. coli. C. sputorum and C. concisus (Tanner et al, 1981), both found in the gingival flora of man, are the principal representatives of the former catalase-negative group of campy lobacters, but division of the genus according to catalase activity (Veron & Chatelain, 1973) is unsatisfactory because of wide variation within single taxa; moreover some strains which give negative catalase reactions have been found to possess intracellular catalase. C. rectus and C. curvus, formerly Wolinella recta and W. curva (Tanner et al., 1981; Tanner, Listgarten & Ebersole, 1984) but transferred to Campylobacter by Vandamme et al., (1991), are also catalase-negative campylobacters found in the mouth. Like C. concisus they are often numerous in gingival and periodontal lesions, although their pathogenicity is uncertain. They are phenotypically and genetically close to C concisus (Paster & Dewhirst, 1988); the only simple distinguishing feature is that they are negative in the conventional oxidase test but positive in the modified test described by Tarrand & Groschel (1982) with the reagent tetramethyl-/?-phenylenediamine (1%) in dimethyl sulphoxide. Because of this we do not include them in Table 7.10c, but they differ from one another in morphology and in sensitivity to certain dyes and antibiotics (Tanner et al. 1984). Nor do we include the genus Wolinella, which was proposed by Tanner et al. (1981) to accommodate these species as well as the bovine rumen organism originally described by Wolin, Wolin & Jacobs (1961) as 'Vibrio succinogenes'. Minidefinition: Campylobacter. Slender helical or curved Gram-negative rods; highly motile by means of a single polar flagel160

[7.10] lum. Coccal forms produced on exposure to air. Optimal oxygen concentration for growth 5-10%; do not usually grow in air or under strictly anaerobic conditions. Capnophilic. Oxidase-positive. Do not ferment or oxidize sugars. Nitrate reduced to nitrite (some exceptions); do not produce indole. Type species: C. fetus; NCTC strain 10842.

7.10.5 Helicobacter (Table 7.10 a, c). The genus Helicobacter (Goodwin et al., 1989) was formed to accommodate the gastric spiral bacterium formerly known as Campylobacter pylori and originally as C pyloridis (Marshall et ah, 1984). Helicobacter pylori commonly colonizes the human stomach mucosa, where it causes superficial 'chronic active' gastritis that probably predisposes to peptic ulceration. Two similar species colonize the stomach of the ferret (H. mustelae) and of cats and dogs (H. felis). Two other species, H. cinaedi and H. fennelliae, formerly Campylobacter cinaedi and C. fennelliae (Totten et al, 1985) were transferred from Campylobacter by Vandamme et al, (1991); they are associated with proctitis in homosexual men, although the natural habitat of H. cinaedi is apparently the intestine of hamsters (Gebhart et al, 1989). H. pylori is rarely isolated from specimens other than fresh gastric mucosal biopsy tissue or brushings. It takes 3-5 days to produce visible growth on primary culture. It is strictly microaerophilic and shares many of the cultural characters of campylobacters, but it produces a powerful urease. It has a single polar tuft of up to 7 sheathed flagella (lophotrichate). Cells undergo rapid coccal transformation (Karmali et al., 1981) and die within one or two hours of exposure to air (Appendix C3.13). Minidefinition: Helicobacter. Helical or curved Gram-negative rods; motile (by means of single or multiple polar sheathed flagella). Coccal forms produced on exposure to air. Optimal oxygen concentration for growth 6%; do not grow in air or under strictly anaerobic conditions. Capnophilic. Catalase- and oxidase-positive. Do not ferment or oxidize sugars. Type species: H. pylori; NCTC strain 11637.

A GROUP OF DIFFICULT ORGANISMS

7.10.6 Arcobacter (Tables 7.10a, c). This genus was designated by Vandamme et al (1991) to accommodate the aerotolerant and low-temperature-growing organism formerly regarded as a campy lobacter, C. cryaerophila (Neill et al, 1985). Arcobacter cryaerophilus is primarily associated with abortion in pigs and cattle, but strains have also been isolated from patients with diarrhoea, mainly children, in developing countries (Tee et al, 1988; Taylor et al, 1989; Kiehlbauch et al, 1991). Several subgroups of A. cryaerophilus are recognized, and species status ('A. butzlerf) has been proposed for one of them (Kiehlbauch et al, 1991). For the present we group all strains under the one species, A. cryaerophilus. Minidefinition: Arcobacter. Helical or curved Gram-negative rods; motile by means of a single polar unsheathed flagellum. Aerobic or anaerobic, but optimal oxygen concentration for growth 3-10%. Growth occurs at 15-37 °C. Oxidase-positive. Do not attack carbohydrates. Nitrate reduced to nitrite. Type species: A. nitrofigilis; NCTC strain 12251. 7.10.7 Anaerobiospirillum (Tables 7.10a, c) is a genus of anaerobic flagellated spiral bacteria with a single species, A. succiniciproducens, first isolated from dogs and described by Davis et al. (1976). Since then strains have been isolated occasionally from human blood and faeces though some of the faecal isolates differ in their carbohydrate fermentation reactions (Malnick et al, 1983; Park et al, 1986; McNeil, Martone & Dowell, 1987). The faecal strains examined in the UK were all isolated on the campylobacter medium of Skirrow (1977) incubated for more than 48 hours in a microaerophilic atmosphere though they required anaerobic conditions for subculture. Anaerobiospirillum is readily distinguished from campylobacters by its negative oxidase and catalase reactions and fermentative activity. We show the essential characters of the genus in Tables 7.10a, c. Minidefinition: Anaerobiospirillum. Spiral, often filamentous, Gram-negative rods; motile; non-sporing. Anaerobic. Catalaseand oxidase-negative. Sugars attacked fermentatively. Type species; A. succiniproducens; NCTC strain 11536.

[7.10] 7.10.8 Streptobacillus (Table 7.10a). A fastidious organism that needs a medium enriched with 20% serum, ascitic fluid or blood, prefers anaerobic conditions for isolation, and for whose growth additional CO2 is needed; it grows slowly even under optimal conditions and growth is inhibited by sodium polyanethol sulphonate (Liquoid) which is frequently used in blood cultures incubated aerobically. It is thus an organism that must be specially looked for if its presence is suspected. Only one species is listed, but it may be but one representative of an almost unknown group of organisms. In morphology Streptobacillus moniliformis is filamentous in young culture but the filaments subsequently break up into shorter sections in which characteristic swellings (but not spores) occur. Biochemical characters may be determined using media enriched with serum and yeast extract by methods that are also suitable for use with L-forms (L-phase variants) of bacteria (Cohen, Wittier & Faber, 1968). The organism is pathogenic for mice, which die within a couple of days of intraperitoneal inoculation; in rats it is a member of the normal flora of the mouth. It is one of the causes of rat-bite fever - with a septicaemic papular rash and arthralgia - in man; outbreaks of infection with this organism associated with milk or water contaminated from rats have also occurred (Shanson et al., 1983). The other cause of rat-bite fever, the spirochaete Spirillum minus, does not usually cause arthralgia. Minidefinition: Streptobacillus. Filamentous Gram-negative rods; non-motile. Nonsporing, but filaments may show swellings. Aerobic and facultatively anaerobic. Needs enriched medium for growth. Catalase-negative; oxidase-negative. Attack sugars fermentatively. Type species: S. moniliformis; NCTC strain 10651. 7.10.9 Legionella (Tables 7.10a, d) represents a genus of motile, pleomorphic, Gram-negative, nonspore-forming rods identified following the outbreak of respiratory infections (Legionnaire's Disease) among those attending a Legion convention in Philadelphia in 1976. There was a high mortality and the causal organism (and type species) was subsequently named Legionella pneumophila (Brenner et 161

Table 7.10d. Second-stage table for Legionella 10 Growth on BCYE* Growth BCYE(-CF)** Growth requirement for cysteine Catalase Browning of BCYE with tyrosine Gelatin liquefied Motility Hippurate hydrolysis Oxidase (3-lactamase Auto-fluorescence 1 2 3 4 5 6 7 8 9 10 11 12

Legionella pneumophila Legionella feeleii Legionella longbeachae Legionella jordanis Legionella oakridgensis Legionella wadsworthii Legionella sainthelensis Legionella micdadei Legionella bozemanii Legionella dumoffii Legionella gormanii Legionella anisa

W = White fluorescence BW = Bluish white fluorescence R = Red fluorescence

d d W 13 14 15 16 17 18 19 20 21 22 23 24 25

11

12

13

14

15

16

17

18

19

20

+ + +

+ + +

+ + +

+ W +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + +

+ + + + + + BW BW BW -

Legionella spiritensis Legionella hackeliae Legionella maceachernii Legionella jamestowniensis Legionella santicrucis Legionella cherrii Legionella steigerwaltii Legionella parisiensis Legionella rubrilucens Legionella erythra Legionella israelensis Legionella tucsonensis Legionella birminghamensis

YG = Yellow-green fluorescence * BCYE = Buffered Charcoal Yeast Extract agar ** BCYE (-CF) = Buffered Charcoal Yeast Extract agar without cysteine and iron

Other symbols used in the table are explained in Tables 5.1 and 5.2 on p.47.

21

22

23

24

25

BW BW BW R

R

-

BW YG

A GROUP OF DIFFICULT ORGANISMS

al, 1979). The genus at present contains more than 30 validly named species (Brenner et al 1985; Brenner, 1986) as well as many unamed though several of them have not yet been isolated from clinical infections and some are represented by only a few strains. They are essentially environmental organisms associated with soil and water (Fliermans et al, 1981) which can grow in suitable niches in waterassociated plant and supply systems, human infection occurring by inhalation of contaminated aerosols. Although not thermophilic, most species (but not L. micdadei) can tolerate elevated temperatures up to 55-60 C for periods of time which destroy most other vegetative bacteria, and many are resistant also to mild acid treatment; these features can be used to advantage for selective isolation, particularly for environmental strains (Edelstain et al., 1982; Dennis, 1988). All Legionella organisms require L-cysteine and possibly iron (in one form or another) in media for primary isolation and most subcultures grow better with them both (Smith, 1982). Buffered Charcoal Yeast Extract (BCYE) agar containing these ingredients as well as a-ketoglutarate and ACES (N-2acetamido-2-aminoethanesulphuronic acid) buffer is recommended for isolation; it can be made more selective by the addition of antibiotics (Edelstein, 1981, 1982). Generic identification either by conventional characterization tests or by the detection of branched-chain fatty acids in the cell wall by gas-liquid chromatography is usually adequate for clinical purposes; for epidemiology, species and, where possible, serotype identification are essential. We show the characters for genus confirmation in Table lAOd; some, but not all, species can also be distinguished by them. Legionellas as a group are relatively inert biochemically and do not attack sugars; a few species, notably L. micdadei, L. longbeachae, L. jordanis and some strains of L. pneumophila, are clearly oxidasepositive, whereas the others give either negative or weak and variable reactions. L. pneumophila is the only current species which consistently hydrolyses hippurate (Herbert, 1981). Most species produce a brown pigment around colonies on Buffered Charcoal Yeast-Extract Agar (BCYE) containing tyrosine (Baine, Rasheed & Feeley, 1978), and the colonies of some species fluoresce in UV light (Weaver & Feely, 1979). There is little evidence,

[7.10] however, to support the proposal (Garrity, Brown & Vickers, 1980; Brown, Garrity & Vickers, 1981) that the latter species should constitute a separate genus, Fluoribacter, or that, based on DNA homology, L. micdadei and related species should form a new genus, Tatlockia, within the family Legionellaceae (Brenner et al., 1985). Species identification depends mainly on fatty acid profiles (Moss et al., 1977) and DNA homology (Thacker et al., 1989) but for routine purposes, differentiation is essentially antigenic and based on serological tests usually by immunofluorescence microscopy; serotypes, biotypes and subtypes can also be distinguished within certain species, notably L. pneumophila (Watkins et al, 1895). A scheme, described by Vesey et al. (1988), utilizing 23 simple tests enabled them to differentiate 21 of 23 recognized Legionella isolates. We show these species in Table 7.10d together with the clinical isolates L. tucsonensis (Thacker et al, 1989) and L birminghamensis (Wilkinson et al, 1987). Other recently named species isolated from environmental sources and defined by DNA homology include L. moravica and L. brunensis (Wilkinson et al, 1988), L. quinlivanii (Benson et al, 1989) and L. gratiana (Bornstein et al, 1989). Detailed information on the laboratory aspects of legionellas, including generic and species identification, is given by Harrison & Taylor (1988). Taxonomically, the genus is firmly established: Legionella is not related to other bacterial genera or to other organisms with a similar (39-43%) G+C content. The division into numerous species is, however, in a state of flux and additions as well as changes are inevitable. It seems likely that genetic differentiation will be essential in future, although we would prefer to see consolidation rather than further splitting. Minidefinition: Legionella. Gram-negative rods; usually motile. Require L-cysteine and iron salts for growth; aerobic but prefer 80-90% humidity with 2-5% CO2. Catalasepositive; do not ferment or oxidize sugars. Type species; L. pneumophila; NCTC strain 11192. 7.10.10 Mycoplasmas (Table 7.10a) are distinct from bacteria and in a class of their own called Mollicutes (Edward & Freundt, 1967). Three genera 163

CHARACTERS OF GRAM-NEGATIVE BACTERIA

are currently recognized: Mycoplasma, which requires cholesterol for growth; Acholeplasma, which does not; and Ureaplasma, which is unique in its ability to hydrolyse urea. Those associated with man include Ureaplasma urealyticum, Acholeplasma laidlawii and several species of the genus Mycoplasma. These organisms may seem out of place in a book devoted to bacteria, but they deserve mention because they may be isolated from clinical specimens as well as from tissue cultures. They may be regarded as Gram-negative because they are decolorized and take up the counterstain feebly. With Giemsa, they stain poorly and the cells are best visualized in broth cultures by dark-field or phase-contrast microscopy. The organisms are extremely pleomorphic and can be difficult to distinguish from artifacts in the medium. The cells do not have rigid walls and they thus resemble the cell-walldeficient L forms of bacteria with which they may be confused. Like L forms, mycoplasmas grow in the presence of antibiotics at concentrations normally regarded as inhibitory for bacteria; unlike L forms, however, they cannot revert subsequently in antibiotic-free media to bacteria with normal cell walls.

164

[7.10]

Mycoplasmas may be found in so-called normal serum and because of their nature and size (0.2 to 0.3 }xm) can pass through some types of bacterial filters; in this way they may thus contaminate media thought to be sterile. However, they are heat-labile so that serum can be made safe for incorporation in media if heated at 56 °C for 30 minutes. Media for the selective isolation of mycoplasmas and suitable for specimens from the genital and respiratory tract have been described (Clyde, 1983Z?; Shepard, 1983; Taylor-Robinson, 1983) but suspect isolates are best referred to a reference laboratory for confirmation and characterization. Like Eikenella corrodens, mycoplasma colonies not only adhere to the surface of the medium but also grow into it and, as the barely visible growth spreads on the surface, the microcolony (10-30 Jim) develops a 'fried' or 'poached' egg appearance. Although not necessarily anaerobic, mycoplasmas grow well in closed environments, benefiting from the trapped moisture content. Individual species are identified by serological growth inhibition tests: the antisera are species-specific and, by using impregnated paper disks, inhibit homologous growth on agar plates (Clyde, 1983a).

8 Taxonomy in theory and practice

As a subject, taxonomy is often regarded as dull and uninspiring. With understanding, however, it can be stimulating and exciting, as indeed were the personal and sometimes unorthodox views expressed by Cowan in the previous edition of this Manual. Moreover, taxonomy is fundamental to the application and use of the increasingly available commercial identification kits now used so widely for medical (and other) bacteria so that knowledge of the basic principles concerned is perhaps more than ever essential. The application of taxonomy, as a science, to bacteriology took a long time. Bulloch's (1938) History of Bacteriology, Brock's (1961) Milestones in Microbiology and Postgate's (1969) Microbes and Man epitomise the applied and practical nature of early bacteriology. This was concerned chiefly with 'diseases' of wine, plants and animals, and little thought was given to theoretical aspects such as taxonomy. The main lines of investigation were to determine causes and, if possible, find cures, from which diagnostic bacteriology and preventive medicine developed. To the early medical bacteriologists, the systematic classification of bacteria was not essential. Indeed it was not until botanists who took an interest in these 'new' and strange organisms tried to apply variations of their own classical rules for naming them that bacterial taxonomy first began. By and large medical bacteriologists were content to let bacterial names look after themselves; they used names which were meaningful to them in relation to illness, such as 'Bacillus typhosus' or more generally 'the typhoid bacillus'. Diagnostic bacteriologists 'named' disease-causing agents in this way so that they could refer to them conveniently again without having to use descriptive summaries; other organisms were discarded as contaminants or commensals.

Cowan referred to the terms classification, nomenclature and identification as the 'Trinity that is Taxonomy'. He likened taxonomy to a cocktail in which these three ingredients were blended so skilfully that they were difficult for non-experts to recognize. Taxonomy describes what a taxonomist does; for bacteriology, it is the theory or science of bacterial classification and its applications. Classification may be defined as the arrangement or distribution of bacteria into orderly groups (or classes) according to common characteristics. Nomenclature provides acceptable rules for the scientific naming or labelling of bacteria according to a standard code of nomenclature. Identification is the assignment of identity to an unknown bacterium with that of a known and named one in a previously made classification. 8.1

Different kinds of classification

As early bacteriologists were concerned essentially with isolation, the first steps in the systematic classification of bacteria were made by botanists, who applied their system of latinized nomenclature. They were followed by increasing numbers of bacteriologists who needed labels or name tags so that they could refer to particular organisms without always having to describe them. Whereas botanists and a few academic bacteriologists classified bacteria along the classical lines of orders, families, genera, and species, practical bacteriologists collected them into groups that were meaningful to those who practised a particular application of bacteriology. Such groupings usually depended then on similarities in what the bacteria did rather than on their physical nature or phylogenetic characteristics, though the latter can now be studied in detail with the development of DNA tech165

TAXONOMY IN THEORY AND PRACTICE niques. Cowan grouped and described the various forms taken by these classifications under the following headings. 8.1.1 Minimal difference classifications. These are subsets extracted from more comprehensive classifications. They form part of the minimal difference identification schemes used by bacteriologists concerned with a limited range of bacteria, often from a particular environment; for example, alkalophilic bacteria, found in highly alkaline pools, require a different classification scheme of their own. Traditional water bacteriology also provides a good example of this type of classification in which coliform organisms are classified according to their morphology, a limited range of sugar reactions and a few biochemical characters. The tests were chosen more from a practical routine laboratory viewpoint rather than because of any special value in species differentiation. The 'water' classification of coliform organisms that developed had little in common with those for enterobacteria but it served a useful purpose for indicating possible contamination of potable water supplies. It is based on a few simple tests, the results of which, together with growth at 44 °C, can be expressed by plus or minus signs using the acronym IMViC (Parr, 1936) for Indole, Methyl Red, Voges Proskaiier and Citrate utilization reactions. Present water bacteriologists may well feel that this is now too simplistic a view as current identification techniques can yield additional information of medical and epidemiological value. 8.1.2 Classification by discipline. Because bacteriology started as an applied science, each discipline developed its own classification of relevant bacteria. With pathologists at the helm, medical bacteriology classified and identified organisms according to the diseases they were thought to cause; other organisms were regarded as contaminants and dismissed as unimportant unless they could be confused with a pathogen in which case they were accorded a place within a scheme of labelled bacteria. Other disciplines took similar attitudes and thus developed along divergent paths. Except between human and veterinary medicine, little exchange of information occurred; thus medical and plant pathologists have worked with similar organisms but, using different 166

[8.1] methods of classification and identification, they failed to realize that the organisms were probably the same, as for example, Erwinia herbicola and Enterobacter agglomerans, and Pseudomonas cepacia and 'Pseudomonas multivorans\ With the increasing need for precise epidemiological studies to pin-point sources of infection, medical bacteriologists gradually began to systematically split the relatively few 'descriptions' of pathogenic organisms into distinct and identifiable bacterial groups. This trend continued with the development first of serological and then of phage and colicine typing methods and ultimately to the present highly specific monoclonal antibody, DNA and other techniques. Much confusion in classification was caused initially by the 'relationships' of bacteria and their association with clinical syndromes. Indeed, until Topley & Wilson's Principles of Bacteriology and Immunity was first published in 1929, medical bacteriologists in the United Kingdom were content to follow the lead of pathologists in considering that bacterial pathogens were related to each other only when the diseases were clinically similar, as with the enteric fevers. For this reason, the pertussis and parapertussis organisms, despite their different test reactions, were originally placed in the same genus, Bordetella. It is interesting to note, however, that DNA studies now support this classification of Bordetella. 8.1.3 All-purpose classifications. In the USA, fewer bacteriologists had their initial training in medicine or in pathology departments and so developments were rather different. Bacteriologists of all disciplines formed the Society of American Bacteriologists - now the American Society for Microbiology - and to them the concept of a hierarchical system for classifying bacteria as described by Winslow et al. (1917, 1920) did not seem so revolutionary as it did to the British. However, the Winslow Committee's reports did not help practical microbiologists to identify cultures and, as Chester's Manual of Determinative Bacteriology (1901) was out of date, the Bergey Committee was formed. Since the first edition in 1923, Bergey's Manual of Determinative Bacteriology has provided both a classification of bacteria and a scheme to help with their identification. Successive editions became a source of invaluable reference material though the hierarchical sys-

DIFFERENT KINDS OF CLASSIFICATION

tern of bacterial classification was abandoned altogether in the last (8th) edition in 1974. The first of four volumes of the current Sergey's Manual of Systematic Bacteriology, comprising essentially Gram-negative bacteria of general, medical, and industrial importance, was published in 1984; the second, for Gram-positive bacteria other than actinomycetes, in 1986; and the last two volumes, for archaebacteria, cyanobacteria and the remaining Gram-negative organisms, and for the actinomycetes, were published in 1989. For students of taxonomy, Sergey's Manuals probably represent the best and most comprehensive of current thought on bacterial classification. 8.1.4 Classifications of convenience. This refers to those kinds of classification which suit the practical bacteriologist and which were exemplified by Rahn (1929). He said that 'It does not matter very much how we divide [bacteria] and which of the many stages of the fluctuating varieties we chose to represent the species, as long as all bacteriologists agree and use the same symbols and names'. But Rahn was a heretic; in 1937 he created the family Enterobacteriaceae with only one genus, Enterobacter, which he did not characterize except by lumping together all the so-called genera that he considered should be included: Escherichia, Salmonella, Aerobacter, Klebsiella, Proteus, Erwinia, Eberthella, Shigella and the gas-formers of Serratia, Pseudomonas, Flavobacterium and Achromobacter, comprising altogether 112 species. This, of course, was even more inclusive than the Enterobacter genus of Hormaeche & Edwards (1960). Apart from introducing the pseudomonads, Rahn's idea was good and deserved much more serious consideration than it got from the bacteriologists of the day. Hierarchical classifications are mainly of theoretical interest whereas identification is a practical exercise. The medical bacteriologist is concerned primarily with day-to-day identification of organisms and not with ranks higher than genus or with any supposed phylogeny; classification is only the means to an end - the hooks on which to hang the bacterial coats. The groupings of bacteria used in this Manual are such hooks: they are for our convenience and do not form part of any formal classification system. The characteristics of the members of the bacterial groups

[8.1] in the Tables represent our choice, and the groupings thus follow from the test characters we have chosen as signposts to the next set of identification tables. 8.1.5 Classification by statistical methods and computer. Statistical appraisal of the correlation of characters (as distinct from the representation, or expression, of characters by numerals) is quite old, as is the principle of attaching equal significance to all characters. Levine (1918) used such a method to classify 333 conform organisms, basing his subdivisions on 13 different characters and excluding those, such as glucose fermentation, that were common to all strains. Little progress was made along these lines until the reappraisal by Sneath (1951 a,b) - succinctly described by Sokal (1965) as 'based on observed characters of taxa rather than on phylogenetic speculation' - led to the development of what is now known as numerical taxonomy. The subsequent developments with this method of classification which, together with that of the computer, have revolutionized bacterial taxonomy - were reviewed by Sneath (1972). Statistical analysis of the characterization data provides an objective, scientific approach which has been used successfully for classifying bacteria from a wide range of sources. The stages necessary for statistical classification involve (a) choice of the bacterial strains and characters to be used; (b) collection and coding of all data in a standard manner; (c) calculation of similarities between strains; and (d) grouping together or clustering the similarities to provide taxonomic groups or taxa. The successful application of numerical taxonomy requires three points to be satisfied: (i) the test characters of a large number of bacterial strains should be determined; (ii) numerous characters should be examined and, as far as possible, these characters should not be subjectively selected. In practice, however, ease of determination makes some characters more popular than others; and (iii) all the characters must be given equal value or weight in the classification. It is not uncommon with modern computers to analyse the characteristics of 300^00 bacterial strains in one project. In practice, with rapid or automated miniaturised laboratory methods, more than 200 phenotypic characterization tests ranging from morphology and physiology to biochemical reactions can be readily performed. 167

[8.1]

TAXONOMY IN THEORY AND PRACTICE It was feared at first that numerical methods might result in considerable splitting and the creation of numerous taxa with but small differences. In contrast, Focht & Lockhart (1965) thought that taxa were more likely to be lumped together by showing up spurious differences in orthodox classifications, perhaps because of imperfectly standardized techniques (Lockhart, 1967). Detailed intra-laboratory studies have demonstrated the considerable extent of innate variation in test results for some widely-used methods when applied to any one species. The objective nature of numerical analyses has shown that the experimental error between tests on replicate cultures of the same strain is about 4-5% when measured over a large number of characteristics. Strict standardization of tests is therefore essential - as it is for all statistical methodology - and any tests that prove difficult to standardize should be discarded. The removal of weighting, however, creates a curious dilemma for the design of diagnostic tables: classifications made by numerical taxonomy do not differ significantly when all sugar reactions except glucose are removed from the numerical analysis. If this is true also for identification, the tables could be much smaller without affecting their usefulness; but like Cowan, we leave this for others to pursue. Once the test reactions have been completed, the results require coding for statistical or numerical analysis. The commonest way is to divide all the test reactions into positive or negative character states (1 and 0 when used with a computer). However, these qualitative results cannot always be used, especially if quantitative tests are included. The process of numerical taxonomy when applied to a large number of test characteristics for numerous bacterial strains requires rigorous statistical methods to assign the extent of similarity between them. The similarities are best expressed as proportions or percentages and the commonest statistical value used in bacteriology is the simple matching coefficient where only those results which match precisely are included in the calculation. Other statistical approaches can also be used for calculating similarities but none should be considered to provide the one and only 'correct' classification. Computers have made this iterative process so easy that all the possible combinations of similarity between the strains can be pre168

sented as a table of similarities or 'similarity matrix'. As the tables are symmetrical, it is usual to express or present them simply as the lower left triangular part of the whole table. One further refinement can be applied to the data if the extent of the dissimilarities between the strains are calculated. These extents represent analogues of distance; classifications can sometimes be expressed in the form of distances between clusters or organisms in an imaginary hyperspace, rather like the astronomic distances between different-sized planets. The taxonomic structure or three-dimensional relationships of the strains can be assessed by carrying out the further stage of cluster analysis or ordination. Again a variety of different statistical approaches have evolved. The two commonest clustering methods are the 'unweighted pair group method with averages' (UPGMA) and the 'single linkage method' (SLM). Principal components analysis with canonical variation is usually used for cluster analysis (ordination) methods so that three dimensional perspective pictures can be presented. The commonest form of representation for cluster analysis is in the shape of a family tree or dendrogram showing the detailed relationships between the various strains analysed. Numerical methods for bacterial classification have tended to either support or disprove the theoretical and tentative conclusions reached by traditional bacteriological methods. However, the lack of objective criteria for assigning ranks to the groups makes it difficult to use numerical taxonomic methods in isolation; those interested in this form of classification should refer to the reviews of Sneath (1972, 1978) for further information. 8.2

Bacterial nomenclature and coding

However good a classification may seem and however easy its units are to define and identify, its usefulness will be tested by its communicability. To be successful the means of communication must be informative and it should be able to reach and be meaningful to those outside the bacteriological field. Communication is made by some form of label, some of which are descriptive and others non-descriptive; some labels consist of words (names, acronyms), others of figures (accession numbers, codes), or of both

BACTERIAL NOMENCLATURE AND CODING

letters and figures (codes). We describe the naming process as nomenclature; when numbers or codes form the label we call it numericlature or coding. Names are the most popular form of label although numbers and coded forms are usually used when more precise information is needed, as for example with phage types and serotypes. Names are often believed to be descriptive (which they need not be) and they can confuse the unwary and mislead the ignorant, like the politician who wanted the generic name Salmonella to be suppressed because he thought it might affect the sales of salmon. Accession numbers (used in culture collections) do not change with the reclassification of organisms, and neither do descriptive codes; both these have a permanency that a name could never hope to attain. 8.2.1 Aims of nomenclature and coding. It is convenient to consider the aims of nomenclature and coding under three subheadings which, in order of importance, are: (i) ease of reference and comprehension, (ii) permanence and stability, and (iii) descriptiveness (meaningfulness). (i) Euphonious words are probably the easiest and briefest form of verbal communication, but words that slip from the tongue of natives of one country may be tongue-twisters to others. Although English is now accepted as the international language for science, Latin - which, as a dead language, has no overt nationalistic overtones - is also used internationally for certain scientific purposes. In speech, however, Latin is far from international: different countries vary widely in the pronunciation of the same spelling. The British, for example, pronounce 'cocci' as 'cockeye' compared with the North Americans who say 'cocks-eye'. For the hard of hearing the difference in sound may render a name quite incomprehensible. Unfortunately, Latin is understood by a decreasing minority of people and we therefore cite the rules which obey those of classical Latin - applicable to Latinized bacterial names. Bacterial binomial species names should be printed in italics (underlined in typescripts) with an initial capital letter for the genus name, which is a latinized substantive noun; the second part of the name, the specific epithet, has an initial lower case first letter (e.g. Escherichia coli). The epithet is a latinized adjective, with the same gender as the genus name. If further ranks, such as sub-

[8.2] species or biotypes, are used, then the trinomial form provides an additional subspecific epithet. This name should also be latinized and follow the same rules as for the specific epithet with an initial lower case letter and the same gender as the genus, as for example, Alcaligenes xylosoxidans subsp. denitrificans. Higher rank names, above genus level, are plural adjectives with a feminine gender, which are used as substantives with initial capital letter. Previously they were not usually printed in italics, but the revised Bacteriological Code (1976) now requires them to be italicised (e.g. Micrococcaceae). Although latinized names should be used on all occasions when scientific accuracy is essential, colloquial or vernacular names are frequently and meaningfully used in verbal communication. We doubt, for example, whether there are many bacteriologists who do not understand the meaning of broad descriptions such as 'coliform organisms' or 'Non-Lactose Fermenters' ('NLF') which embrace many different species in a perspective wider than that of genus. (ii) Permanence usually applies to bacterial labels only if they are descriptive. When the label is a word that is part of a classification (as Pseudomonadaceae derived from Pseudomonas) it may change when there is a change in classification. All bacteriologists understand that classifications, like clothes, can alter with increased knowledge and fashion, and changes in fashion may be accompanied by changes in name. (iii) Descriptive labels do not usually change with revision of a classification; the organism that causes enteric fever has exactly the same characteristics whether it is called the typhoid bacillus, 'Bacillus typhosus\ "Bacterium typhosum\ 'Eberthella typhosa\ or Salmonella typhi. If it were described by a code, as say 'species 12345', that label could be permanently attached to it. However, numerical coding schemes are neither popular nor practical for global use, though conversely they are eminently suitable for culture collections. Why should bacterial names change? Mention has already been made of reclassification based on increased scientific knowledge following taxonomic study. This may result, for example, in better definition of the members of a genus so that new species or re-naming of old species may follow; thus Proteus morganii is now known as Morganella morganii after it was shown to be sufficiently different, taxonomi169

TAXONOMY IN THEORY AND PRACTICE cally, from the other members of the genus Proteus. However, changes in fashion are far less frequent since the publication of the Approved Lists of Bacterial Names (1980). In addition, changes in name do not usually mean changes in identifying characteristics. There is one other method by which a name may be changed: it is also far less frequent now, and involves the principle of priority of publication. If a prior and validly published scientific description of an organism is found to exist with a different name, then this older name may be used to replace the current one. The Approved Lists have been accepted as the starting date for future reference and they record all the bacterial names that were both recognizable and in current use on 1st January, 1980. Bacterial species were listed only if they had been adequately and validly described and, where cultivable, if a type, neotype or reference strain was available. A type strain is intended to be the most typical strain chosen to represent and characterize a species so that other (unknown) strains can be compared with it for classification or identification purposes. Type strains sometimes become lost and new strains (neotypes) are selected to replace them. Reference strains are often derived from these two categories but are used to represent a whole array of similar strains in comparative work. Any names not included in the Approved Lists of Bacterial Names in 1980 automatically became 'without standing' although provisions exist in the International Code of Nomenclature of Bacteria (1976 revision) for their revival if an exceptional case is shown to be valid. Any subsequent changes or additions are published either as papers or, from time to time, in validation lists in the International Journal of Systematic Bacteriology (USB). These lists have thankfully put an end to the previous necessity of searching interminably through ever-increasing mountains of literature merely to determine the earliest name used for a taxon in order to make a valid new addition to bacterial nomenclature. Since the starting date in 1980, new names must be published or announced in the International Journal of Systematic Bacteriology, which is currently the only publication officially recognized for this purpose. Summaries of the validated additions and changes in nomenclature are published annually in the IJSB. In addition, it is intended to publish compendia of all 170

[8.2] such changes at longer intervals, the first of which is the index by Moore, Cato & Moore (1985) which lists all names published in the IJSB or other journals and validated by the Judicial Commission from 1980 to 1985. 8.2.2 Bacteriological codes. All scientific names of bacteria are governed by the International Code of Nomenclature of Bacteria, colloquially called the 'Bacteriological Code'. After initial proposals (Lapage et al., 1973), it was revised and published on behalf of the International Committee on Systematic Bacteriology by Lapage et al. in 1975; however, as it only became effective on 1st January 1976, it is therefore usually referred to as the 'Bacteriological Code, 1976 revision'. The Bacteriological Code, which was originally based on botanical nomenclature, was first published in 1948 with the aim of providing a set of rules to promulgate some stability, if not fixity, among bacterial names and to avoid confusion by providing a sound basis for systematic bacteriology. It contained precise rules for naming bacteria as well as a judicial procedure for settling disputes about them. However, it was subsequently amended repeatedly and eventually contained so many appendices that it became too complex and unwieldy. Although Cowan (1970a; 1971) did not have too much time for such rules - regarding many of them as pedantic - he did much to promote the climate which led to their revision in 1975. This revision greatly simplified the principles of the Bacteriological Code, which he regarded as sound. Although much of it is still devoted to rules governing the spelling of names, which Cowan considered far less important than clear and adequate descriptions of organisms, the revised Bacteriological Code has in turn led to the acceptance and application in 1980 of the Approved Lists of Bacterial Names. We suspect that Cowan would have given his blessing to these lists though he might well have taken exception to a few of the names. Together, we think that the Bacteriological Code and the Approved Lists of Bacterial Names will provide a firm basis for classification and nomenclature in the future. We would not do justice to Cowan if we omitted to say that he took exception to Principle 5 of the Bacteriological Code. This concerns the nomenclatural type strain as the means for attaching a name to a

BACTERIAL NOMENCLATURE AND CODING

taxon. It states that The application of the names of taxa is determined by means of nomenclatural types', referred to as 'types'. The designation of such types, whether of species, genera or other taxa, is governed by rules that make it clear that the nomenclatural type is not necessarily the most typical or representative element of any taxon. Cowan, however, in this Manual (1974) pointed out that ideally it was an obvious requirement that the strain chosen to represent a species should actually be characteristic of that species. Indeed, he agreed with Gordon (1967) who opposed the designation of a single (type) strain to represent a species. Her concept of a species was that of a microbial population which she defined as 'a group of freshly isolated strains, of strains maintained in vitro for many years, and of their variants, that have in common a set of reliable characteristics separating them from other groups of strains'. Cowan also agreed with Gordon's view (which he knew was not generally accepted) that the properties of strains persisting after years of cultivation in the laboratory were useful criteria for defining the species. 8.3

Bacterial identification

An unknown organism cannot be identified as a member of a particular group until that group or taxon has been described, named and established. In this Manual we do not consider methods for identifying medical bacteria which cannot be performed in routine clinical laboratories. It should be noted, however, that DNA-DNA hybridization, cell wall composition, fatty acid profiles and other techniques may help to characterize and thus assist in the better classification of a group of strains, though they are of limited use to the bench-oriented users of this Manual. Strict standardization and reliability are essential guidelines for good identification methods. Accurate and clear descriptions of test methods are essential for their communication to and satisfactory establishment in other laboratories. Vague information such as 'positive for utilization of citrate' should play no part in diagnostic test descriptions; the medium , its composition, the method, the time and temperature of incubation and the reading and interpretation of the result all add definition to the basic statement. The demand for speed is sometimes an excuse for poor

[8.2] technique in the laboratory or for the use of rapid but crude methods. Rapid methods, however, are not inherently erroneous provided they give reliable results. It is only when rapidity leads to shoddy workmanship and thus error in identification that they cannot be tolerated. For reliable characterization, pure cultures must always be the starting point for any identification method. This rules out any method in which the inoculum for a set of tests is a colony taken from a primary plate of a selective medium which suppresses but does not kill unwanted organisms. The routine use of a hand lens to assess purity is an important part of good microbiological practice. Similarly, use of the straight wire (and a plate microscope) when subculturing separate colonies for purity from a mixture is likely to give rise to fewer problems than the wire loop. Identification has been described variously as the lowest form of taxonomic work or as classification in reverse; Mayr (1968) characterized taxonomy as 'essentially the technique of identification'. Identification consists of characterizing the unknown strain as much as is practicable, and comparing its characters with those of known, classified and named organisms. When they match, the unknown strain will be identified with a known organism. If the number of characters compared is small then a 'perfect' match will be relatively common; but more often, when many characters are compared, a near match with a small number of discrepancies is much more likely than true identity. For reliable identification,we have already considered the choice of bacterial characters and the methods for ascertaining them; in the following sections, we describe briefly the various ways of recording and comparing the characters. 8.3.1 Identification by diagnostic tables. Conn (1900) used tables with +, ± and - signs for the bacterial characters of known organisms. The characters of unknown strains were recorded on slips of paper and moved over the table to obtain the best visual match; indeed Cowan & Steel (1961) used Perspex strips in much the same way. However, Conn and his colleagues (1907) later commented that 'unless the characteristics of species can be clearly and distinctly tabulated, it is almost a hopeless task...to identify a new culture with one previously described.' None the less tables of charac171

TAXONOMY IN THEORY AND PRACTICE ters, especially sugar reactions, became fashionable though the symbols varied and were often more complex than necessary even for a simple positive result. Subsequent attempts to combine two characters such as acid and gas production in one symbol eventually resulted in tables too complicated for use and, failing standardization of symbols, they fell into disrepute. Tables returned to favour again when a simple, standardized listing arrangement for each organism together with clear descriptions of characters was introduced in the reports of the Enterobacteriaceae Subcommittee (Report, \954aM 1958). These lists of characters could be combined and they were used by Cowan (19566) and by Cowan & Steel (1961) in their early work on diagnostic tables which led subsequently to this Manual. To be of any use, diagnostic tables must have clear symbols to denote the various possible results. Despite pressure, we have continued to use symbols rather than percentages in diagnostic tables for this Manual because the latter, although perhaps more accurate, are much more difficult to compare and interpret quickly. The main disadvantage of tables, especially if large, is the difficulty in appreciating and interpreting the significance of occasional mismatches with the test reactions of unknown strains. 8.3.2 Dichotomous or diagnostic keys. Botanists use diagnostic keys extensively, but they have never been popular with clinical bacteriologists, who prefer an established laboratory routine that can be followed for most organisms isolated from specimens. An attempt to use dichotomous keys commercially for identifying the Enterobacteriaceae in the early 1970s was soon abandoned with recourse to tabular formats. Indeed the dichotomous keys in Bergey's Manual of Determinative Bacteriology were of little practical help until that of Skerman (1949) was added to the seventh (1957) edition. The main disadvantages lie in the exact nature of the result. Each test has to be done in the correct order and the next test to be performed depends strictly on the result of the previous test. Such keys are most successful when the characters used are stable and give constant, clear-cut results. Their usefulness therefore varies with different kinds of organisms: Streptomyces species for example as well as other higher taxa can be identified readily 172

[8.3] using them (Kuster, 1972) but they are unlikely to be used in practice to identify enterobacteria. 8.3.3 Mechanical devices. Cowan described these as the various 'bits and pieces' and 'Heath Robinson' gadgets - such as the 'Determinator' of Cowan & Steel (1960, 1961) - created as an aid to simplify the identification of bacteria. They all suffer from the same disadvantages as dichotomous keys in that they cannot allow enough leeway for the exceptions that constantly occur among biological material. They have now largely given way to computerized assistance, though mechanical devices were developed to help with the intricacies of profile construction for API and similar products (see Section 10.5). 8.3.4 Identification by cards (polyclaves). With polyclaves or multiple keys, any character can be chosen at random and further characters can be investigated in any order. Identification is made by excluding all but the final answer. Polyclaves are well suited to punched cards, called the 'Peek-a-boo' system, in which cards of a uniform size are notched at the periphery for each taxon. The position for each notch denotes a specific test result: notched for positive, no notch for negative. To be practicable, card systems must be based on the most useful distinguishing characters of organisms and they can only be as good as the tables or other information on which they are based. Like the diagnostic tables in this Manual, punched cards are suitable, with or without computer sorting, for use with a progressive identification system in which a 'minimal difference' set of cards first identifies the genus (or main group) and other sets (or packs) then identifies the species. If not used in a step-by-step system, the cards for the character information would need to be large with numerous holes; to be sufficiently comprehensive, such cards would be too clumsy for routine use. In the previous edition of this Manual, Cowan described in detail a practical and successful punched card system for identifying to both generic and species level bacterial strains encountered in medical and veterinary laboratories. We reproduce this 'Identicard' system with minor changes in Chapter 9 and Appendix E in this third edition as an aid for laboratories where computer assistance may not be read-

BACTERIAL IDENTIFICATION

ily available. The data still hold good though a few modifications have been made in line with recent taxonomic changes. The methodology involved has also been updated and computerized by Feltham, Wood & Sneath. (1984) and is available commercially as a computer program; it includes assistance with finding the relevant bacterial identification tables in the present third edition of this Manual and also the relevant pages in the 7th edition of Bergey 's Manual of Determinative Bacteriology. 8.3.5 Arithmetical summation. Fey (1959) devised an identification system in which each bacterial character was scored with a unique but weighted number: for example, acid from mannitol = 5; gas from glucose = 10; growth in KCN = 320, etc. The scores of all the positive characters of the unknown organism were then added together to give a total score which would correspond to one of the scores in a table for organisms of known identity. In practice, the scheme was not reliable as any test discrepancies resulted in a wrong score with consequent misidentification (Steel, 1962a; Baer & Washington, 1972). As for any identification system, this simply confirmed that standardization of tests with adequate controls is absolutely essential; given this, it has potential though with the advent of commercial identification kits and computerized methods it is unlikely to be used. 8.4

Bacterial taxonomy in the future

The recent application of more objective methods of identification, especially numerical taxonomy, as well as the development of quantitative techniques for the measurement of genetic relatedness and evolutionary divergence among bacteria have impinged on traditional classification and nomenclature as used in this Manual, sometimes with untoward consequences. For example, the name Pasteurella pestis for the plague organism was well known but there was confusion about its status as a pathogen when it was renamed after transfer to the genus Yersinia (Williams, 1983). Review of the various approaches to bacterial systematics in the light of this newer taxonomic understanding was therefore important and an ad hoc committee of the International Committee

[8.3] on Systematic Bacteriology met in 1987 to examine the wider implications and consider how the different approaches to bacterial systematics could be reconciled with each other. As its report (Wayne et aL, 1987) is of considerable importance for taxonomy in the future, we discuss briefly the essential points and recommendations here; the full report is reproduced with permission in Appendix I. The committee based its discussions on four aspects of bacterial systematics: (i) Phylogenetic studies, which are directed towards understanding the evolutionary paths which taxa have taken from early and more recent ancestors. These pathways can be discerned from analyses and comparison of the evolutionary distances between certain 'semantides': large genetic information-bearing molecules such as nucleic acids and proteins, (ii) Descriptive studies, which act as a link between the semantide-based phylogenetic taxonomy and traditional bacterial systematics in which organisms are described in phenotypic terms as in this Manual. Such descriptions help to define taxonomic groups of organisms which may also be recognised in phylogenetic terms, (iii) Diagnostic perspective, where a selection of phenotypic features or of 'probes' derived from phylogenetic analysis are used to recognize unknown strains and assign labels or names to them. Such names, mostly of genera, species and subspecies, are those familiar to readers of this Manual, (iv) Associative work, in which vernacular names are used to evoke practical information about strains, such as their medical, industrial or ecological significance. The committee suggested that a single all-embracing hierarchical scheme should be adequate for the taxonomy of all bacteria provided vernacular groupings are kept for diagnostic purposes. The complete DNA sequence would represent the ultimate ideal reference standard for determining phylogeny, which in turn would determine taxonomy; at present DNA reassociation is the nearest approach to this ideal for species. Taxonomic schemes based on phylogeny must, however, exhibit phenotypic consistency. Hierarchical consistency is also important though the actual demarcation points in RNA dendrograms will vary with different branches and can be influenced strongly by stable phenotypic characterizations. Formal nomenclature should agree with and reflect the genomic relationships and is essential for major 173

TAXONOMY IN THEORY AND PRACTICE groupings such as genera which form the basis of bacterial systematics. It was recognized, however, that there is at present no satisfactory phylogenetic definition of the term genus and that consequently, as with higher taxa, flexibility in approach is necessary because the actual scope of the definitions of different genera will undoubtedly vary. For families, chemotaxonomic and hybridization or sequence data should be consistent with each other. Species currently represent the only taxonomic units that can be defined in phylogenetic terms. Such phylogenetic species would include bacterial strains with about 70% or more DNA-DNA relatedness at the optimum temperature for reassociation and less than 5% divergence of unpaired bases within related sequences. Phenotypic characters should accord with these criteria and would be allowed to override this phylogenetic concept of species only in exceptional instances. The committee recommend that distinct genospecies that cannot be differentiated from others by any known phenotypic character should not be named until they can be clearly distinguished in this way. The designation of subspecies is, however, acceptable for organisms that are closely related genetically but which have different phenotypic characters. The nomenclature for species should reflect genomic relationships to the greatest extent possible except where to do so rigidly might create a 'nomen periculosum' which, though taxonomically correct, could cause uncertainty, confusion or danger in practice. For example, the name Yersinia tuberculosis subsp. pestis was proposed instead of Yersinia pestis

174

[8.4] for the plague organism to reflect its close phylogenetic relationship with the species Yersinia tuberculosis. This was regarded as a 'nomen periculosum" and rejected because it might cause serious confusion in clinical medicine; the correct name accordingly remains Yersinia pestis. Similar reasoning was applied to retention of the species and name Bacillus anthracis for the anthrax organism instead of accepting it as a pathogenic variety of Bacillus cereus. Further recommendations are made about other concerns including the special difficulties expected in the taxonomic reorganization of the major phylogenetic taxon referred to as 'the purple photosynthetic bacteria and their relatives'; overcoming the problems inherent in the growth and identification of nonor hardly cultivable bacteria; and the status of organisms only cultivable in ecosystems. The report of the ad hoc committee embodies much of the philosophy and most of the practicalities which Cowan enunciated in this Manual in his own inimical style. The recommendations, which we endorse, go a considerable way towards reconciling the divergent scientific approaches to bacterial systematics. We hope they will perhaps stem the tide of new bacterial names - many based purely on phylogenetic characters - which have been appearing since the Bacteriological Code (1976 revision) and Approved Lists of Bacterial Names (1980) were promulgated. Indeed, the report reinforces the approach to traditional bacterial systematics as used in this Manual.

Bacterial identification by cards

Identification of bacteria is not always an art. The process of recording, collating and interpreting their common diagnostic characters can be mechanized and used in a visual sorter (Olds, 1966, 1970) based on the Peek-a-boo system (Wildhack & Stern, 1958; Yourassowsky et al., 1965). Hand-sorted punched cards have been used for recording quite varied taxonomic information (Wood, 1957) and, in the first edition of this Manual, Cowan & Steel suggested that the 'tables could form the basis of a set of diagnostic punched cards to be used with similar cards on which the characteristics of the unknown [isolates] are punched'. Soon after that was written, Schneierson & Amsterdam (1964) described a punched card used for identifying bacteria at the Mount Sinai Hospital, New York, but unfortunately they did not give details of the 'authoritative reference' sources from which they obtained the characters for their master cards - information which is of course essential for assembling and using tables of characters. A disadvantage of diagnostic tables is that as more and more detail is included they become less easy to use. For the second edition of this Manual, the early drafts of what became Tables 6.1 and 7.1 stretched to more than twenty columns with up to ten lines of characters; to check them Cowan made a set of punched cards with one (or more) cards for each genus. Checks with these punched cards revealed that some of the columns in the tables were in fact similar - though this had not been noticed previously - thus enabling them to be combined and the tables rearranged. Though laborious, this exercise demonstrated how quickly a bacterial identification could be made once the characters had been notched on a card. A new and perhaps better way of using the diagnostic tables was thus confirmed.

9.1 Minimal-difference Identicards for genera For the identification of bacterial genera encountered in medical and veterinary laboratories, only eleven holes are needed; an additional spare space (hole) can be used also for a favourite or supplementary character that is regarded as useful. Cards are punched as described in Appendix E. A corner is cut off each card so that thet can all be faced correctly for notching. Pre-punched cards available commercially can also be used. The characters used must include the Gram reaction, which is not shown in the tables; the others comprise the ten used in Table 6.1, which also cover those in Table 7.1 as well as many of those mentioned in the minidefinitions given in Chapters 6 and 7. The name of the genus is placed in the middle of each master card, together with any relevant information helpful for isolation, subculture (e.g. oxygen requirements of Campylobacter or Eikenella) or identification. Some genera need multiple master cards. For example, Aerococcus, in which the catalase test may be read as either weakly positive or as frankly negative; Clostridium, in which C. perfringens is nonmotile as well as reluctant to spore; Gemella, so easily decolorized, has a card for both the Gram-positive and Gram-negative states; and Nocardia is shown separately as acid-fast and as not acid-fast. The enterobacteria are dealt with as one genus but three master cards are needed for the identification of shigellas, including Shiga's bacillus (Shigella dysenteriae 1), which is unusual in being catalase-negative. The notching of the individual master cards is illustrated in Fig. 9.1, which also shows the kind of supplementary information that can be entered in the central area of the card. 175

[9.1]

BACTERIAL IDENTIFICATION BY CARDS

Table 9.1. Aggregations of genera that may occur with the genus Identicard set Aggregation

Genera (species) in aggregation

(i)

Aerococcus (catalase read as negative) Gemella Pediococcus Streptococcus Enterococcus

(ii)

Actinomyces Bifidobacterium Clostridium perfringens (not sporing) Eubacterium

(iii)

Arachnia Erysipelothrix Lactobacillus

(iv)

Mycobacterium Nocardia

(v)

Leuconostoc Peptostreptococcus

(vi)

Enterobacteria (Shigella dysenteriae 1) Streptobacillus

(vii)

Bordetella Brucella Moraxella

(viii)

Actinobacillus Pasteurella Aeromonas salmonicida

(ix)

Aeromonas Chromobacterium violaceum Plesiomonas Vibrio

To use the Identicard set for genera, first examine the unknown culture for the generic characters used on the card. The acid-fastness and the sporing ability need not be determined for every strain, and it would be surprising if every isolate was tested for anaerobic growth unless the culture failed to grow aerobically at the first attempt. Positive characters of the unknown organism are notched from the appropriate hole of a blank card. Rod-shaped organisms (bacilli, vibrios) are assumed unless hole 5 (coccus) is notched; alternatively, for an actual record for every character, additional holes may be used and notched. 9.1.1 Aggregation of cards. In many instances the characters chosen will be sufficient to identify to genus level most of the bacteria likely to be isolated in medical and veterinary laboratories; however, 176

because so few generic characters are used in marking up (notching) the cards, some unknown strains will inevitably end up after sorting with more than one card, and the genus identification must then be made by studying the characters given in the secondstage (or third-stage) tables of Chapters 6 and 7, or from using further sets of Identicards (see Section 9.2). The most surprising aggregation of genus Identicards occurred with Shigella dysenteriae 1 and Streptobacillus (Table 9.1, no.vi) though in practice there would be no difficulty in distinguishing them at the bench; the similarity simply begins and ends with the characters chosen for Table 7.1. In contrast, the other aggregations are of similar organisms such as Actinobacillus and Pasteurella (sensu stricto) that are difficult to separate even by experienced bacteriologists. 9.2

Identicards for species

The user of the Identicard set for genera will probably find species identification easy by consulting the tables of Chapters 6 and 7, but where the tables are large a further set of species Identicards may be helpful. The notching of the master cards for species is complicated by the presence of many 'dee' (d and D) characters and by late reactions recorded in the tables within round brackets. These difficulties are overcome by having two rows of holes in the master cards (Fig. 9.2a); characters recorded by + signs in the tables are notched to the second (inner or plus) row of holes, and characters for different or delayed reactions shown by d, (d), or (+) are notched only to the first (outer or dee) row (Fig. 9.2b). As an example, the notching of the master cards for the species identification of enterobacteria is shown in Table E5 in Appendix E. Positive characters of the unknown strain to be identified are notched to the inner row of holes (Fig. 9.2c); alternatively special cards with one row of holes in line with the inner row of the master set may be used. 9.2.1 Aggregates of species cards. With salmonellas, aggregation of species is to be expected in the Identicard set as the differences between most members of the group are essentially serological, and such differences are not entered on the cards. To a

[9.2]

IDENTICARDS FOR SPECIES

/

o o2

3

O 4

o5 6O

7O 12

11

o o

10

O

9

O

8

O

1 = Gram reaction 2 = Motility 3 = Acid-fastness 4 = Spore formation 5 = Coccus (rod is not notched) 6 = Growth anaerobically 7 = Growth in air 8 = Oxidase 9 = Catalase 10 = Optional 11= Sugars attacked oxidatively 12 = Sugars attacked fermentatively

Fig. 9.1. Schematic example of card punched with a single row of holes illustrating notched cut-out for a positive [+] character. Used for master cards (characters of known genera) and as cards for recording the characters of an unknown organism for genus identification. lesser extent, aggregation applies also to the cards for the Shigella group, which Bascomb et al. (1973) found difficult to identify by probabilistic methods. 9.2.2 Variants. As the Identicards are based on characters recorded in the tables of Chapters 6 and 7, they are subject to the same limitations. Like the tables, they do not make provision for aberrant strains or the varieties that may occur, sometimes limited to a particular locality: the so-called 'local races'. It should always be borne in mind that a non-motile organism may be a non-motile variant of a motile species, or it may represent technical failure to detect motility. In both motility media and broth cultures, motility is generally most active in cultures incubated below the optimal temperature for growth of the

organism; a temperature between 22 and 25 °C is satisfactory for most mesophilic bacteria. Anaerogenic variants of Salmonella serotypes which normally produce gas may occur and make identification difficult; they can even lead to suspicion that a new 'species' has been isolated. Reactions that are slow or delayed, as for example gelatin liquefaction by Enterobacter cloacae, or lactose fermentation by Shigella sonnei, are notched to the first (outer) row of the two-row master cards; by so doing and combining it with the method of probing cards recommended in Appendix E (Tables E5, 6), a reading made before the character has developed (or the test become positive) will not lead to misidentification.

177

[9.2]

BACTERIAL IDENTIFICATION BY CARDS

(a)

o o o o o o

/

/ /

OO

oo oo oo

20

1

2

o o o o

3

4

outer (dee) row inner (plus) row

\J *•

6

TO

19

»O

18

9O

17 16

15

14

13

o o o o o o

12

11

io O

o o o o o o

d (d)

+

(+)

oo oo oo oo

5

("} < vj * /-\ <

o U o o o o Master card

/ /

o o o o o °/I

oo oo oo oo

o o o o

o o u o/ Card for unknown

)

]

I

o O O1 o o o/

Fig. 9.2(a) Card punched with two rows of holes: inner (plus) row for positive (+) characters; outer (dee) row for variable [d, (d) or (+)] characters i.e. different reactions with different strains: positive with some, others negative. Used for master cards (characters of known species) in sets for species identification. May be used as cards for characters of unknown organisms. (b) Master card, showing the method for notching d, (d), (+) and + characters. (c) Card of unknown organism (to be identified) notched for a positive character.

178

10 Bacterial identification by computer

In 1968, Dybowski & Franklin suggested various theoretical models for using computers to help with the identification of bacteria, but the first practical systems for computer-assisted identification were not introduced until 1973 by Friedman et al. and Lapage et al. They depended on a full set of standardized routine tests carried out under strict laboratory control. One such system for strains difficult to identify in diagnostic laboratories has been perpetuated in the Identification Services Laboratory in the National Collection of Type Cultures (NCTC) at Colindale, London. The work of this laboratory was extensively reviewed by Willcox, Lapage & Holmes (1980). Other national culture collections have set up similar services for their own countries. The ranges of the tests applied have increased progressively in order to accommodate new species and genera. Bacterial groups which have been updated recently or examined in this way include the aerobic fermenting and non-fermenting Gram-negative rods (Holmes & Dawson, 1983; Holmes, Pinning & Dawson, 1986), the Gram-positive aerobic cocci (Feltham & Sneath, 1982) and the genus Bacillus (Logan & Berkeley, 1984). Other bacterial groups have been investigated elsewhere. Commercial kit suppliers have constructed large databases for their particular range of tests and organisms. As with the Tables in this Manual, the test reactions for identification databases or matrices have often been compiled from a variety of sources. The tables or matrices consist of columns usually showing the percentage of positive test reactions, each row representing a cluster or group of similar organisms. These clusters usually equate to named bacterial species. Each entry in the matrix should be the best estimate for a positive result for that test with that particular group of organisms. This estimate may be

obtained from data in the literature which can be combined with the laboratory results given by sample strains and isolates to provide a subjective percentage of positive reactions for the matrix. A more objective database is obtained by aggregating test results for representative strains from each group of organisms with those of recognised type or reference strains. The validity of each entry in the table thus depends on the examination of adequate collections of strains by accepted and suitably controlled tests: the importance of these aspects cannot be over-emphasized. 10.1

Strain collections

The best databases for bacterial identification are those generated from the actual results for particular groups of organisms associated with particular ecological niches and examined by reproducible differentiation tests. Bacterial isolates from patients must be used to construct databases for use in medical laboratories; bacteria from water, soil and fish, for example, should only be used to assist with the identification of aquatic and environmental organisms. As the majority of commercial identification systems currently available have been produced for medical laboratory needs, the tests and databases may thus prove unsuitable for bacteria from other habitats. Many of the best bacterial identification tables stem from numerical taxonomic studies where the Adansonian concept of large numbers of unweighted tests provide objective clustering of strains. Data from other sources, such as DNA homology and chemotaxonomy, provide additional evidence to help place strains within defined clusters. The entries in published identification matrices show the probabilities of a positive test reaction expressed as a range usually from 0.01 to 0.99 or 179

BACTERIAL IDENTIFICATION BY COMPUTER

alternatively as percentages from 1 to 99%. To be exact, if a test is always negative for a particular bacterial group, the figure should be a probability or percentage of 0 but, to compensate for any conceivable error in the method and to accomodate the single, exceptional isolate that disproves the rule, a slightly greater figure (0.01 or 1%) is used. For example, urease-positive strains of Escherichia coli do occur, albeit uncommonly, so that an entry of 0% would be erroneous. To compensate for this modification at the bottom of the scale, a similar adjustment is made at the top end so that entries of 0.99 or 99% are used instead of 1 or 100%. 10.2

Test selection

There is no magic formula for assessing the 'best' tests or the 'right' number for any specified bacterial group. The NCTC uses approximately 60 conventional tests in their scheme whereas commercial kits rarely use more than 24. The 'best' tests are those which are most easily determined and which have a high degree of stability and resistance to operator error. Test reactions can vary depending, for example, on the size of inoculum, the duration and temperature of incubation, the composition and viscosity of the medium and the 'eye of the beholder'. Errors can and do occur when recording results if the end-point is not clearly defined. For example, in the Hugh & Leifson test for Fermentation or Oxidation, the viscosity of the paraffin layer is as important as the definition of the yellow colour produced by acid from the breakdown of glucose. Is the reaction positive if only the top 10 mm is yellow? And was the fermentation tube heated and cooled before inoculation to drive out traces of oxygen? For reasons such as these, computer-assisted identification techniques must be defined precisely and with great care. Indeed colour charts for the end-point of such reactions are extremely useful for their standardization. Variation in test results can be caused also by the selection of characters liable to phage- or plasmid-mediated changes. Such tests would obviously be unsuitable for identification purposes as the variable results would affect the level of identification. The selection of tests for identification databases

180

[10.1]

must rely on quantifiable reactions which can be used to determine the percentage of positive results. Tests such as pigmentation, colony size or degree of acidfastness are thus not easily accommodated in computer-assisted identification tables or databases. The quality of a database can be assessed subjectively by testing a range of known strains. As mentioned already, a number of conditions can affect the database quality but one of the less-defined effects can be caused by geographical variation. Strains isolated in a particular country or even in a region within a country may be phenotypically different from other strains of the same name elsewhere. As examples, some strains of Yersinia enterocolitica are virtually continent-specific; and Serratia strains are common isolates from urine in the Bordeaux region of France, but exceedingly uncommon in the UK. Sneath (1979«, b\ 1980a, b, c) and Langham et al. (1989) have developed a range of objective techniques for assessing the quality of identification databases by means of computer programs designed either to analyse the degree of spatial overlap of neighbouring imaginary hyperspherical clusters or to rank the characters in order of their separation power. The best tests are those which separate the taxa within the database into two equal parts whereas the worst cannot differentiate between any taxa. Other programs are used to analyse the database for the most typical identification likelihood score for each entry using a range of statistical identification procedures, including taxonomic distance, described in Section 10.4. These techniques were developed to assess the practicality and correctness of particular published databases without the need to obtain strains and test them against the tabulated values. We would advise those who wish to construct their own databases to obtain copies of these programs and ensure that their databases satisfy all the criteria specified. 10.3

Identification of an unknown isolate

The identification of an unknown strain relies on comparison of its test reactions in turn with those of the known species in each column, or test, in the table. This comparison can be quantified in terms of the relationship of the reaction pattern of the

[10.3]

IDENTIFICATION OF AN UNKNOWN ISOLATE

unknown strain with that of the statistical nearest pattern among the rows, or species, in the table. If the unknown strain matches a particular species to a defined level, it will identify with that species. If the unknown strain does not match well with any tabulated species, then it would be classified as unidentified with that database. This might be because (i) the database was poorly defined, (ii) operator error occurred in the handling of the unknown strain (e.g. use of an impure culture or unsuitable conditions for growth), (iii) the unknown strain should not have been tested against that particular database but rather a different one: for example, a non-fermenting organism would not identify well with a 'fermenter' database. 10.4

programs such as those published by Sneath (1979a). Consider strains in four hypothetical bacterial groups of taxa (ONE to FOUR) each examined by six characterization tests (A to F). Suppose that the numbers of positive results for each test with each taxon are expressed as percentages, as follows: Table 10.1. Percentage of positive test reactions. Test A

B D E C Percentage of positive reactions

F

99 99 1 1

1 99 99 1

90 1 1 50

Taxon ONE TWO THREE FOUR

99 99 1 1

99 1 90 10

50 1 99 85

Identification: statistical principles

The statistical process in identification involves calculation of the likelihood or closeness of match of all the characters of the unknown strain with those of each row or species in the relevant table or database; this ensures some degree of ranking or ordering of the identification scores. The principles of many of the various statistical methods which have been applied were described by Sneath (1978); the actual mathematical calculations of those usually used are given in detail by Willcox, Lapage & Holmes, (1980). The commonest approach relies on a taxonradius model with accumulation of probabilities so that the unknown strain can be compared with a group of imaginary hyperspheres in multi-dimensional space, each test character providing one dimension in that space. The bacterial groups (rows or species) in the table may be perceived as imaginary, hyperspherical clusters separated spatially from other clusters. Each cluster may be defined statistically with a centre of gravity (centroid) and a critical radius that includes a high proportion of the bacterial strains within that group or taxon. Thus each row in an identification table can be defined as a hypersphere with a given position or centroid and radius. The probabilistic likelihood statistic used involves multiplication of the probabilities that the test reactions of the unknown strain match those for any particular row or species in the table. A hypothetical example will perhaps best illustrate the method which can readily be used with straightforward computer

These percentages can be expressed as probabilities of positive test reactions and the results given by an unknown strain, examined by the same tests, entered thus: Table 10.2. Probabilities of positive test reactions and example for an unknown strain. Test

ONE TWO THREE FOUR

F

A

B C D E Probability of positive reactions

0.99 0.99 0.01 0.01

0.01 0.99 0.99 0.01

0.99 0.99 0.01 0.01

0.99 0.01 0.90 0.10

0.50 0.01 0.99 0.85

+

-

+

+ Not Tested

Taxon

UNKNOWN -

0.90 0.01 0.01 0.50

The test reactions of the unknown strain are essential for the calculation of the Likelihood scores for matching a particular taxon. To calculate the Likelihood scores, the probabilities of the reactions are multiplied together for each taxon for each test result given by the unknown organism. Any reactions that were doubtful or not tested may be left out of the calculation. If the unknown strain gives a positive test reaction, the actual probability is used, whereas if the reaction is negative, then 1 minus the taxon probability is used to give Likelihood score values thus: 181

[10.4]

BACTERIAL IDENTIFICATION BY COMPUTER

Table 10.3. Likelihood score values for the unknown strain. Taxon

Likelihood scores for unknown strain

ONE TWO THREE FOUR

(1-0.99) x 0.01 x (1-0.99) x 0.99 x 0.50 (1-0.99) x 0.99 x (1-0.99) x 0.01 x 0.01 (1-0.01) x 0.99 x (1-0.01) x 0.90 x 0.99 (1-0.01) x 0.01 x ( l - 0 . 0 1 ) x 0.10x0.85

= = = =

Sum of all Likelihoods

= 0.8653699

4.95E"7* 9.89E"9 0.864536 8.33E"4

*E = Exponential; thus 4.95E" 7 = 0.000000495

Normalization or standardization of these Likelihood score values is then necessary to provide a true comparison and to compensate for the effects of using 0.01 and 0.99 (or 1% and 99%) for the lower and upper entries in the table. This is achieved by dividing each Likelihood score in turn by the sum of the Likelihood scores for all the taxa, which gives Taxon THREE the high score of 0.999 instead of an apparently mediocre score of 0.864536: Table 10.4. Normalized likelihood scores for the unknown strain. Taxon

Normalized Likelihood score for unknown strain

ONE TWO THREE FOUR

4.95E"7 + 0.8653699 9.89E-9 - 0.8653699 0.864536 -s- 0.8653699 8.33E^ + 0.8653699

= 5.72E-7 = 1.14E-8 = 0.999037

As an alternative to the normalization procedure for finding the nearest match or 'best fit', the Most Typical Likelihood value for each row or taxon giving the most likely result for each reaction may be determined. This is calculated as follows: Table 10.5. Most typical likelihood scores for the unknown strain. Taxon

Most Typical Likelihood scores for unknown strain

ONE TWO THREE FOUR

0.99 x (1- 0.01) x 0.99 x 0.99 x 0.5 0.99 x 0.99 x 0.99 x (1- 0.01) x (1- 0.01) (1- 0.01) x 0.99 x (1- 0.01) x 0.9 x 0.99 (1- 0.01) x (1- 0.01) x (1- 0.01) x (1- 0.1) x 0.85

182

= 0.480298 = 0.950990 = 0.864536 = 0.742279

Instead of using the normalization procedure, the previous Likelihood score values calculated for the unknown strain may be divided by these Most Typical Likelihood scores for each taxon to give Modal Likelihood scores for the unknown strain thus: Table 10.6. Modal likelihood scores for the unknown strain. Taxon

Modal Likelihood score for unknown strain

ONE TWO THREE FOUR

4.95E"7 + 0.480298 9.89E"9 -0.950990 0.864536 + 0.864536 8.33E"4 + 0.742279

= = = =

1.03E"6 1.04E"8 1.00000 1.12E"3

From these Modal Likelihood scores it is now apparent that the unknown strain matches completely with taxon THREE, whereas with the normalization procedure the Likelihood score was lower although it was still the highest. For the identification of unknown strains, both of these statistical techniques are frequently used either alone or together with other variants and data such as estimates of the frequency of occurrence of each taxon in the database (Willcox, Lapage & Holmes, 1980). 10.5

Profile numbers

Direct access to computers with relevant identification programs is not always necessary for identifying an unknown strain by means of these statistical methods. Lists of the most commonly occurring patterns of results which have been processed by specially written computer programs can be obtained from commercial sources : these patterns of test reactions are compared with the relevant database table by means of the commercial computer program which uses one of the two statistical identification techniques described to calculate identification scores (Stevens, 1980). Other relevant information such as serology or additional tests can also be appended to the presumptive identity for better differentiation. Test results for given organisms can be aggregated to give unique profile numbers which denote the particular patterns of positive and negative test results. To form such profiles, the characterization tests are arranged in groups of three (triplets) and positive

[10.5]

PROFILE NUMBERS

results score either 4, 2 or 1 in each triplet of tests as follows: Table 10.7. Profile numbers from 0-7 for triplets of tests. Result given by Profile digit

Test 1

Test 2

Test 3

Test score

The various combinations of positive and negative test reactions yield a total of eight different patterns of triplet results which may be fully described by unique 'octal' set of single profile digits from 0 to 7 with profile scores of 4, 2 and 1. The majority of commercial and home-produced test kits use this octal format, though API uses the reverse by scoring 1, 2 and 4. The full Bacterial Identification Profile (BIP) is constructed from the individual profile digits for each set of three tests: thus 21 tests give a 7 digit profile. If there are less than three tests in the last group of tests, only the corresponding scores are

used; for two tests, 4 and 2 are used for the positive results; and for one test, only 4 or 0 can be scored. Bacterial Identification Profiles are thus a very concise way to express many results precisely but briefly and conveniently. The profiles can be listed in numerical order to form indices or registers of characterized organisms. Each entry can simply have the name of a specific organism attached to it or, in fuller registers, an abbreviated identification calculation with scores together with the names and scores of two or three of the closest possible species. Further information is usually provided about useful additional tests which could be carried out to yield a better identification score as well as on any tests which have given unexpected results and so count against a better identification score. Commercial firms are not unique in producing lists of identification profiles for common bacteria. This can be done in routine diagnostic laboratories as exemplified by Clayton et ah (1986), who generated such a profile list from the work described in their paper for the routine identification of Gram-negative rods by using a multi-point inoculation method. The methods described briefly in this Chapter illustrate the basic principles concerned with the data collation and recovery necessary for current 'expert' bacterial identification systems (Valder et ai, 1987; Gavini et ai, 1990). Such methods will probably be used increasingly in the future as computer hardware becomes more powerful and software more complex.

183

11 Quality control in microbiology

From the beginning, this Manual has preached consistently not only about the importance of pure cultures for identification but also of the need for standardization of test methods both within and between laboratories. We reinforce these views here with a brief outline of the current concepts of quality control and laboratory proficiency which must be understood and put into practice for good identification procedures to succeed. As before, we emphasize the importance of controlling characterization tests with organisms known to give positive and negative results; the test organisms recommended are given in Appendix D together with media and conditions suitable for their propagation short-term and storage. 11.1

Laboratory quality control

Quality control can be regarded as the continual monitoring of equipment, reagents and working practices as well as the provision of training with specified details of laboratory methods and procedures to ensure proficiency. The lack of test reproducibility both within and between laboratories, discussed in previous editions of this Manual, has now been well documented (Sneath & Johnson, 1972; Lapage et al., 1973; Sneath, 1974; Sneath & Collins, 1974; Snell, DeMello & Phua, 1986). Variation in test results between laboratories may be due to differences in the sensitivities of the methods used. Such differences are of considerable practical importance as the results given in the diagnostic tables and keys may be applicable only if the methods stated are used. This applies also to micromethods and diagnostic kits. Variation in test results within a laboratory may be caused by several factors and can result in misidentification if the test in question is a key one in a limited set. The prime aim of quality control for bacterial characterization tests is therefore to elimi184

nate or reduce to a minimum the extent of test variation both within and between laboratories. In a comprehensive approach to laboratory quality control, several factors must be considered, including the standardization of procedures; provision of a laboratory 'manual' of detailed written methods; stock control of media; purity checks on test strains and on media for contaminants; record keeping; and checks of test procedures and controls with known strains. 11.1.1 Standardization of methods is essential to reduce test variation to a minimum. One quality control survey, for example, revealed 21 methods or variations of them for testing for bacterial motility. In general, laboratories use particular methods either because of long experience and confirmed reliability, or with some, simply because of inertia. In the latter case, the methods must be critically evaluated. Details such as inoculum size, time and temperature of incubation, composition of media and the ingredients used including the particular brands can all affect some test results. All such details must be standardized within each laboratory. 11.1.2 A laboratory manual containing full details of all methods and procedures used in a laboratory is essential to avoid the drift which inevitably occurs when methods are passed verbally from one person to another. Such a compendium or manual should be written by staff with many years of practical experience in order to be authoritative, and it should be kept up to date. The instructions should relate to what is expected in practice rather than to a theoretical ideal and they should be reviewed regularly. The methods should be readily available for daily use and reference within every laboratory, so more than one copy is necessary.

LABORATORY QUALITY CONTROL 11.1.3 Stock control and shelf life are not easy to define and quantify but some general points will suffice to illustrate the necessary requirements. An estimate of the likely shelf life of media is valuable. A turnover period of about one calendar month is a reasonable compromise for most characterization media. Storage space, batch size and preparation times can be planned accordingly. After this period, unused media should be discarded to prevent accumulation of older batches. This permits the date marking of batches of media rather than of individual tubes or bottles. The temperature of storage must be cool and evaporation should be prevented by using either sealed plastic bags or Bijoux bottles, Universal containers or the like with screw caps. Humidity can cause deterioration of dehydrated products, especially if hygroscopic. If stored in the refrigerator, such products should be allowed to reach room temperature before opening to prevent the condensation of water vapour with subsequent hydration of the material. Long-term storage media may be necessary for those used for tests performed infrequently: in cool conditions some media can be stored in sealed tubes for up to 6 months. However, these media must be checked with known positive and negative control strains each time they are used. Media or reagents not suitable for extended storage times include for example V factor discs, ONPG broth, Selenite broth, hydrogen peroxide and some carbohydrate media. Ingredients, such as peptones, which are liable to deteriorate, should be purchased in small quantities and the bottles marked with the dates of receipt and opening. They should be discarded if there is any evidence of change caused by moisture. 11.1.4 Purity checks for contaminants should be carried out on all batches of freshly prepared media. Gross contamination will usually be apparent after 5 days at room temperature. Media should be incubated also at the temperature used for the test before each batch is released. Media for characterization tests should be inoculated only with cultures known to be pure. Subculture directly from selective media significantly increases the risk of mixed growths and, despite the delay, it is safer to follow the procedures for purity described in Section 3.2.

[H.l] 11.1.5 Simple record-keeping not only provides assurance that quality control procedures have been carried out, but enables faulty batches to be identified and discarded, encourages a methodical approach and helps to emphasize the concept of accountability. 11.1.6 Test procedures and controls. All characterization tests should be controlled with organisms known to give clear-cut positive or negative results under appropriate conditions. The suitability of both media and reagents should be checked with known test strains immediately after preparation as well as during any period of extended storage. Misreading of test results due to lack of familiarity is an important cause of error and control organisms are useful for instructing staff in the correct appearance of such reactions. For these purposes, lists of the species and strains recommended are given in Appendix D. Many laboratories maintain small culture collections of their own. Such stock collections, especially of pathogens encountered infrequently, are invaluable both for teaching purposes and for comparison with field isolates. Strict safety precautions must be observed with such laboratory strains and the description 'teaching organisms' must not be taken to mean 'harmless' (Blaser et aL, 1980). For the long-term storage of laboratory strains, several procedures are available. Freeze-drying is the method of choice as it virtually ensures long-term stability of characters and freedom from contamination with simple storage conditions for the ampoules. The necessary apparatus is, however, expensive for small laboratories and as an alternative the simple method of drying described by Rhodes (1950) which needs only a vacuum pump and phosphorous pentoxide can still be used. Storage in liquid nitrogen (-196 °C) guarantees survival and stability but the nitrogen requires constant replenishment. Most medically-important bacteria can be stored satisfactorily in Glycerol Broth at temperatures below -20 °C (typically -70 °C) for several years (Feltham et aL, 1978; Jones et al., 1984). Desiccation of bacterial strains on gelatin discs also provides a convenient storage method (Snell, 1984a). Regular subculturing is the least suitable method because of the potential instability of test characters and the possibility of contamination (Snell, 1984&). Frequentlyused strains may be kept on nutrient media for convenience but at the end of their storage life (Table D4) 185

QUALITY CONTROL IN MICROBIOLOGY

they should be replaced from stock cultures preserved by one of the more secure long-term storage methods (e.g. -70 °C or freeze drying). A more comprehensive list is given by Lapage, Shelton & Mitchell, (1970). Cooked meat is a good maintenance medium for many organisms, including anaerobes. Dorset egg is also a good general purpose storage medium; salmonellas retain their antigenic structure well on it though shigellas become rough. In general, media that are less nutrient-rich are better for keeping cultures than richer media in which they die out more quickly. Media containing fermentable carbohydrates should be avoided for maintaining cultures and selective media should never be used. Cultures must not be allowed to dry out and tightly closed screw-capped containers are preferable to waxed or corked tubes. 11.2

Laboratory proficiency and quality assessment

Quality assessment, known also as proficiency testing in some countries, acts as a check on the efficiency of quality control procedures by the introduction of simulated and occasionally real specimens of known but undisclosed content into the laboratory for examination and reporting. This may be done internally within the laboratory and/or externally but in either case the specimens should be examined by the same staff that would normally examine similar clinical material. When organized internally, specimens of known content are introduced into the routine system by senior staff who intercept and evaluate the reports (Estevez, 1980). This offers the advantage that assessment specimens can be more easily 'disguised' as part of the routine intake and thus offer a more realistic appraisal of the standard of work. With external assessment, an organizing laboratory sends specimens of known and undisclosed content to participating laboratories for examination and receives the completed reports for analysis. These specimens are more difficult to camouflage and laboratory staff tend to give them special treatment. However, an external assessment scheme offers several advantages including stability of the specimens, the availability of repeat samples in cases of failure and the opportunity to compare individual performance with the general standard of other participants. National and regional external quality assessment 186

[H.l] schemes for microbiology have been developed and now exist in several countries. For clinical material national assessment schemes are perhaps preferable to international schemes because the microbes encountered, the reporting and referral practices, as well as the methods and antimicrobial agents used will vary between countries. International assessment schemes are, however, important for harmonisation as well as for public health and regulatory control purposes, especially for perishable produce imported or exported for human or animal consumption. For those considering implementing a national monitoring service we describe briefly the United Kingdom National External Quality Assessment Scheme for microbiology (UKNEQAS) administered by the Public Health Laboratory Service (PHLS). The scheme is comprehensive and is not limited simply to the isolation and identification of bacterial pathogens but includes viruses, fungi and parasites. It embraces diagnostic microscopy and serology as well as antibiotic assays. The working principles were described by Snell, DeMello & Gardner (1982) and Snell (1984c). On joining the scheme, each participating laboratory is given a unique code number by which it is identified in all transactions so as to maintain confidentiality. Simulated clinical specimens are prepared in the Quality Assurance Laboratory, Division of Microbiological Reagents and Quality Control (DMRQC) at the PHLS Central Public Health Laboratory (CPHL), Colindale, London. All specimens are distributed to participants together with 'request' details and 'report' forms. After examination of the specimens, each participating laboratory reports its findings to the organizing DMRQC laboratory following which a brief outline of the intended results is issued. The reports are then analysed and all participants receive a summary of the overall results for each distribution and a computer-derived analysis of individual results on current as well as recent specimens. Twelve distributions, each of three specimens, for assessing isolation and identification procedures are issued each year. Participation is free to all health care microbiology laboratories in the UK and almost 500 laboratories are included. Although the great majority of specimens are straightforward and contain bacteria likely to be found in clinical practice in the UK, more unusual specimens are occasionally distributed for education-

LABORATORY PROFICIENCY AND QUALITY ASSESSMENT

al purposes or where recognition of an unusual pathogen - such as Corynebacterium diphtheriae or Vibrio cholerae - could be of concern for public health or to persons from abroad. The proportion of 'positive' material is obviously higher than in normal clinical practice. Specimens may contain fully virulent pathogens other than those regarded as highly dangerous in Hazard Group 4 (Advisory Committee, 1990) so that due regard must be paid to safety and hygiene in the laboratory. Detailed information on the preparation of such specimens is given by DeMello & Snell (1985). Freeze-dried mixtures of bacteria are usually used as simulated specimens. They are chosen to provide a realistic challenge to media and technical skill in the isolation and identification of pathogens from the mixed flora associated with the clinical site in question. Rigorous quality control tests on the specimens are carried out by DMRQC with checks before, during and after the distribution including the return of duplicate specimens through the post from various geographically dispersed laboratories. If the quality control checks reveal unexpected and unacceptable changes in the content of a specimen, it is regarded as void and the participating laboratories are informed. In such instances, the results are not included in the calculation of laboratory performance statistics. Three weeks are allowed between distribution of the specimens and return of the final reports on them from the participating laboratories. After this period repeat samples are available for those laboratories which did not obtain the expected result with the original specimen. The performance statistics distributed to each laboratory include scores for the most recent distribution and an analysis of performance ratings over an inclusive six-month period. This gives details of the specimens supplied, the number of specimens reported as 'not examined', the number of reports not returned and the number of reports received too late for analysis. The analysis gives the total laboratory score, derived by addition of the individual scores for each specimen, the total possible score and the average score. The participating laboratory can use this information for direct comparison with other laboratories and can see whether their performance is better or worse than the average. Laboratories can thus use the

[11.2]

data to monitor their own performance and to take any action required if problems are revealed. Confidential help and advice to participants with continuing problems is available from a national Advisory Panel for Microbiology. Most participants appear to find the UKNEQAS useful and good relationships are maintained, perhaps because the scheme is seen as educational rather than as regulatory. 11.3

Laboratory safety and hygiene

We include a few elementary precepts on safety and hygiene in the microbiology laboratory in order to reinforce their importance even in taxonomic work. As well as general and statutory considerations concerning safety in the laboratory, particular care and a high standard of personal hygiene are essential in microbiological work because pathogenic organisms may always be encountered. In addition, media not necessarily designed for pathogens may support their growth so that great care is needed in the handling and disposal of all cultures. Sound technique is the basis of safe microbiological procedures and it is important that all individuals concerned with microbiological work should have received adequate training. It is also important that the necessary laboratory equipment and facilities should conform to accepted codes of safety and good laboratory practice (Advisory Committee, 1991). Certain precautions are essential in the microbiology laboratory not only to prevent contamination of samples and culture media but also to avoid infection hazards to personnel: - observe strict personal hygiene; keep nails short; and use hair and beard protection if necessary; - wash hands with soap and warm water both before and after microbiological work as well as after use of the toilet; - always wear protective clothing in the laboratory; - sterilize all cultures by autoclaving, and sterilize or disinfect associated apparatus and materials before disposal or reuse; - do not eat, drink or smoke in the laboratory; - report any accident, spillage or unusual occurrence to senior staff; - if in doubt, ask for advice. 187

APPENDIX A Preparation and control of culture media Al General considerations A2 Formulae of media A3 Media control

Al

GENERAL CONSIDERATIONS

The ability of some bacteria to grow on media that are inhibitory to other bacteria gives the former organisms additional special characteristics that may help in their identification. The media themselves, with their selective and differential qualities, are of particular interest in diagnostic bacteriology and for this reason we have sometimes included a few notes on the isolation of certain bacteria and have referred occasionally to enrichment and other media used in culturing organisms from clinical material. We emphasize, however, that this Manual is concerned with how to identify organisms that have been isolated and not with how to isolate them. In this appendix, media are listed under the following section numbers and headings: A2.1 Basic media A2.2 Enriched and enrichment media A2.3 Differential and selective media A2.4 Media for enhancing pigment production A2.5 Media for carbohydrate studies A2.6 Miscellaneous media A 1.1

Cleaning and sterilization of glassware

Glassware for media such as Koser' s citrate, in which there is a single source of an element such as carbon or nitrogen must be chemically clean to be free from it. A recommended method of ensuring this is to boil all tubes in 20% nitric acid for 5-10 minutes and then wash and rinse well with with glass-distilled water. Tubes are dried in an oven in the inverted position in baskets lined with filter or blotting paper to prevent the mouths of the tubes touching the metal. Glassware such as Petri dishes and test tubes with metal caps or cotton-wool plugs are sterilized in a hot air oven, preferably with a fan, at 160-170 °C for one hour. The efficiency of the oven and the even distribution of heat should be checked each day by placing Browne's green spot type III indicator tubes on each shelf (Darmady, Hughes & Jones, 1958; Brown & Ridout, 1960). Overheating of tubes plugged with cotton-wool must be avoided; several workers

188

(Wright, 1934a; Drea, 1942; Pollock, 1948) have drawn attention to the inhibitory effect of substances volatilized from cotton during dry heat sterilization. Tubes and flasks capped with polypropylene covers (Varney, 1961) and screw-capped bottles (with caps loose) are sterilized by autoclaving. A1.2

Indicators

The pH indicators used in bacteriology are shown in Table Al; some, such as phenolphthalein, are not readily soluble in water and are dissolved in dilute alkali or ethanol. The preparation of Andrade's indicator and litmus solution are more complex and details are therefore given in Sections Al.2.1 andAl.2.2. A 1.2.1

Andrade's indicator (modified from Andrade, 1906) Acid fuchsin 5g Distilled water 1000 ml N-NaOH (as required) 150-180 ml

Dissolve the acid fuchsin in the distilled water and add 150 ml of alkali solution. Mix and allow to stand at room temperature with frequent shaking for 24 h; the colour should change from red to brown. If the dye has not been sufficiently decolorized add a further 10 ml of alkali solution, mix thoroughly and leave for another 24 h. Subsequent additions of alkali may have to be made until a straw-yellow colour is ultimately attained with the minimum of alkali. To test the final indicator, add 1 % of the indicator solution to Peptone Water at pH 7.2, mix thoroughly and determine the pH; note and record the rise in pH due to the alkalinity of the indicator. Should for example the increase in pH be 0.2, all batches of medium to which this batch of indicator is to be added must have an initial pH value 0.2 lower than the desired final reaction. * Albert Browne Ltd., Chancery House, Abbey Gate, Leicester LE4 0AA, UK.

CULTURE MEDIA Table Al. Indicators and their characteristics

Indicator

Usual concentration (%)

ml 0.05 N-NaOH per g indicator

Solvent

pH range

Colour change (acid to alkaline)

Methyl red Chlorphenol red Andrade's Litmus Bromcresol purple Bromthymol blue Neutral red Phenol red Cresol red Thymol blue Phenolphthalein

0.2 0.2 (See A 1.2.1) (See Al.2.2) 0.2 0.2 0.1 0.2 0.2 0.2 0.1

_ 47 37 } 32 57 , 53 43 J -

50% ethanol water or 50% ethanol water 40% ethanol water or 50% ethanol 50% ethanol water or 50% ethanol 50% ethanol

4.2-6.3 4.8-6.4 5-8 5-8 5.2-6.8 6.0-7.6 6.8-8.0 6.8-8.4 7.2-8.8 8.0-9.6 8.3-10.0

red-yellow yellow-purple pink-yellow red-blue yellow-purple yellow-blue red-yellow yellow-red yellow-red yellow-blue colourless-red

Al.2.2 Litmus solution (modified from Mclntosh, 1920) Litmus, granular 250g Ethanol (40%) 1000ml Grind the litmus and place in a flask with 500 ml of the ethanol; boil for 1 min, decant and keep the liquid; add the remainder of the ethanol to the residue, boil for 1 min, decant and add this liquid to that retained. Centrifuge and adjust the volume of the supernatant to 1000 ml with ethanol (40%); add N-HC1 drop by drop until the solution becomes purple. To test for the correct reaction, boil 10 ml of distilled water, cool, and add one drop of litmus solution; after mixing the water should become mauve in colour. This indicator solution is used at a concentration of about 2.5%.

hot-water jacketed funnel is used; alternatively, use a Buchner funnel and filter rapidly through paper of greater wet strength. A method for the filtration of agar-containing media during sterilization in an autoclave was described by Brown (1961). Gelatin-containing media used to be clarified with the aid of egg-white or horse serum; gelatin for bacteriological use is now of such quality that this step is unnecessary and gelatin media may be clarified by the same methods as those containing agar.

A 1.3

A 1.6

Carbohydrates

'Sugars' used in bacteriology are shown in Table A2, where they are grouped by their chemical structure. A 1.4

Clarification of media

Liquid media may be clarified by filtration through paper, sintered or fibre glass, or, if still available, asbestos fibre (Seitz filtration). To prevent adsorption of any constituents, it is better to avoid the use of a filter aid such as kieselguhr or talc. Wright (\934b) found that filtration of broth through a thick filtering layer resulted in marked reduction in its growth-promoting capacity. The grades of material suitable for clarification of media are shown in Table A3. Agar-containing media must be clarified while in the molten state; for this purpose use paper pulp or a grade of filter paper especially designed for the filtration of agar sols. To prevent solidification during filtration, a steam or

A1.5

Sterilization of media

The method of sterilization recommended for each medium is included with the details of preparation. Storage of media

Although freshly prepared media are usually desirable for the cultivation of most bacteria, with some, such as Wilson and Blair's (1927, 1931) Bismuth Sulphite Agar, maturation or 'ageing' is necessary for satisfactory selectivity; nor should egg-based media be used new. Many media can be safely kept at room temperature or in a refrigerator for several weeks, or even months, before use. Stability varies with the individual medium and it is not possible to fix a universally applicable storage life. The important point is that moisture should be retained. To prevent evaporation and concentration of the constituents during storage, media should be kept in screw-capped rather than in cottonplugged containers. Appreciable evaporation can occur in a refrigerator but this can be prevented by putting tubed media and plates in polythene bags. If nutrient agar slopes appear to be dry, they should be melted and re-solidifed though not on more than one or two occasions. Strong light is detrimental to most media and storage in

189

APPENDIX A Table A2. Carbohydrates used in bacteriology

Class

Carbohydrate

Convenient concentrations in water (w/v) at 20 °C for stock solutions

Pentoses

Arabinose (L+) Xylose Rhamnose (/sodulcitol) Fructose (laevulose) Galactose Glucose (dextrose) Mannose Sorbose Cellobiose Lactose Maltose Melibiose Sucrose (saccharose) Trehalose Melezitose Raffinose Glycogen Inulin Starch, soluble Aesculin (esculin) Amygdalin Arbutin Salicin Adonitol Dulcitol Erythritol Glycerol Mannitol Sorbitol Inositol

40 50 40 50 30 50 50 40 10 15 50 10 50 25 10 10 5; dissolves to produce an opalescent solution 20 at 70 °C; solution may be slightly opalescent - soluble in hot water 0.1; 7.5 on heating 7.5 10 3 40 5; readily soluble on heating 40 miscible in all proportions 15 50 15

Methyl pentose Hexoses

Disaccharides

Trisaccharides Polysaccharides

Glycosides

Alcohols

Non-carbohydrate

Table A3. Filtration materials, and grades, suitable for clarifying media Agar-containing media

Papers: Green's Whatman Postlip Schleicher & Schiill Delta Munktell Sintered glass: Jena Pyrex Asbestos pads (if available): Seitz Sterimats Carlson

190

1 )

Liquid media

Simple funnel

Buchner funnel

798 72; 904 7 3 l;30;52 633E 520B 73; 598 7 3 3173A 5

500 72; 904 73 15 agar-agar 520A 72; 520B '/2

960;993 52 agar-agar 520B 376 0

' K3; K4; K5 FCB K3;K5

CULTURE MEDIA Table A4. Usual volumes of media (in ml) for tubes of various sizes Tube size Medium

75 x 12 mm (3 x 72 in)

100 x 12 mm (4 x 7 2 in)

125 x 12 mm (5 x 7 2 in)

150 x 16 mm (6 x 5/s in)

Liquids Slopes (slants) Slopes with butts Stabs

1-2 1 2.5 2.5

2-2.5 1.5-2 2.5 2.5

3 2.5-3 3.5^ 4

4-5 5 7 7

Table A5. Concentration (%) of agar in media for different purposes Agar Type of Medium

Japanese

New Zealand

Ionagar no. 2*

American

Solid (normal) Solid (to inhibit spreaders) Semisolid 'Motility media'

1.5-2.0 7 0.1-0.5

1.0-1.2 4 0.05-0.3

1.0

1.5

0.7

0.3-0.4 0.1-0.4

* Ionagar no. 2 was a special purpose agar used only when a defined medium was needed, It has been replaced by Purified Agar (Oxoid L28).

the dark is preferable, especially for those containing dyes or indicators. Egg media should be kept for a long period (e.g. two weeks) before use in order to give contaminants that grow only at room temperature the opportunity to develop into visible colonies. Cotton-plugged tubes are a potential source of contamination as the moisture absorbed even by 'non-absorbent' cotton wool is sufficient to permit development of fungi, and hyphae may penetrate a cotton plug. Media that have been kept in a refrigerator should be allowed to attain room temperature before use. Because the solubility of gases in liquids decreases with increase in temperature, it is essential to check that gas bubbles do not appear in the inverted inner (Durham) tubes of carbohydrate media as they warm up from cold storage to room temperature. Liquid media to be used for anaerobic cultivation (particularly those containing thioglycollate) should be heated in a boiling water bath to remove dissolved air and allowed to cool undisturbed before use. Poured plates may be kept at 4-15 °C, butthey should not be put close to the cooling unit of a domestic-type refrigerator. Excess water of condensation can be avoided by allowing the medium to cool to about 55 °C before pouring it into Petri dishes. If plates are to be stored in an

exposed position they should be stacked with the lid uppermost; in canisters they should be inverted. Plates for anaerobic use may be pre-reduced by storing in an anaerobic atmosphere either in jars or in a cabinet. Higher forms of life, including insects, beetles and mites, can also be troublesome in a laboratory. Indeed, 'footprints' left by mites crossing agar media inoculated with cultures have been observed on plates kept on the bench or incubated at 22 °C and in medical laboratories this is potentially dangerous. Dehydrated media are particularly liable to insect attack; the eggs laid by adults hatch into larvae which can then feed upon carbohydrate material. A 1.7

Volumes of media

The volumes of media suitable for dispensing into Petri dishes vary with the size of the dish. For routine bacteriological purposes in the United Kingdom, there is a current British Standard specification (BS 611) for glass Petri dishes (1978) and for those made from 'plastics' (1990) though the former is about to be withdrawn as obsolete. Media volumes of 15-20 ml are recommended for these dishes which have an internal diameter of 90 mm (3.5 in). Volumes suitable for media in tubes are shown in Table A4. 191

APPENDIX A A2

FORMULAE OF MEDIA

Two terms which refer to all formulae need explanaion. Agar. In this Appendix the agar concentration * elates to Japanese agar; when New Zealand agar is used the amount stated in the formulae should be multiplied by 0.6 to give approximately the same gel strength. Concentrations of agar for different types of gel are shown in Table A5. Agar is made from seaweed from many parts of the world but the origin is seldom stated on the label of commercial preparations; consequently Table A5 and others like it are not as useful as they appear. Regard should be paid to the information supplied by the manufacturer and the concentrations advised for different purposes. Agar is a poor conductor of heat (Bridson & Brecker, 1970) and time must be allowed for heat penetration both in media making and for safety purposes to ensure that cultures on agar plates are properly sterilized before disposal. The altitude of a laboratory may affect the ease with which agar goes into solution and it may not be practicable to dissolve agar in a steamer; above 2000-3000 feet, autoclaving at about 0.36 kgf/cm2 (5 lbf/in2) may be essential for melting agar. Water. The term water, used in all formulae, implies potable tap water. When distilled water is specified in a formula, de-ionized water may be used. A2.1

Basic media

A2.1.1

Nutrient Broth

Meat (beef) extract 10g 10 g Peptone NaCl 5g Water 1000 ml Dissolve the ingredients by heating in the water. Adjust to pH 8.0-8.4 with 10 N-NaOH and boil for 10 min. Filter while hot through thick filter paper to remove phosphates which are precipitated in alkaline solution at this stage. Adjust to pH 1.2-1 A, and sterilize at 115 °C for 20 min. Notes. Nutrient Agar is nutrient broth gelled by the addition of 2% agar. Semisolid Nutrient Agar (Craigie agar, sloppy agar, slush agar) is Nutrient Broth containing 0.4% shredded agar. Double-strength Nutrient Agar follows the same formula as nutrient agar but the volume of water is reduced to 500 ml. Layered plates consist of two layers of solidified medium; in layered Blood Agar (Section A2.2.1) the bottom layer consists of Blood Agar base, Peptone Water Agar (Section A2.1.2) or Saline Agar (Section A2.1.11) and the top layer is Blood Agar base with 5% blood added. The thin top layer may contain other substances under

192

test; some, such as chitin, are insoluble and the suspension is made either in Water Agar (Section A2.1.12) or Saline Agar (Section A2.1.11). A2.1.2 Peptone Water Peptone 10 g NaCl 5g Water 1000 ml Dissolve the solids by heating in the water. Adjust to pH 8.0-8.4 and boil for 10 min. Filter, adjust to pH 1.2-1.4, and sterilize at 115 °C for 20 min. Peptone Water Agar is Peptone Water gelled by the addition of 2% agar. A2.1.3

Robertson's Cooked Meat medium (modified from Lepper & Martin, 1929) Minced meat 1000 g 0.05 N-NaOH 1000 ml Add the minced meat to the alkali solution, mix well and heat to boiling; simmer for 20 min with frequent stirring. Skim off the fat and check the pH, which should be about 7.5. Strain through gauze or muslin, squeeze out excess liquor, and dry the meat particles at a temperature below 50 °C. For use, place sufficient dried meat in a screwcapped container to a depth of about 2.5 cm and add sufficient Nutrient Broth to give a depth of about 5 cm. Sterilize at 115 °C for 20 min; avoid rapid release of pressure in the autoclave after sterilization. Note. Although called Robertson's Cooked Meat medium, the method of preparation differs considerably from the original description (Robertson, 1916). Cooked Meat medium (alternative formula) Horse meat, fat-free and minced 450 g Distilled water 1000 ml Sodium thioglycollate, 45% aq. soln 1 ml NaCl 5g Peptone 10 g Boil the meat in the water for 1 h. Filter through muslin and press the meat dry. Add the NaCl and peptone to the filtrate, adjust to pH 8.4 with 10 N-NaOH and bring to the boil. Filter and add the thioglycollate solution. Distribute the dry meat particles to a depth of 2.5 cm into screw-capped bottles, add the prepared broth to a level of 5 cm. Sterilize at 115 °C for 20 min; allow the pressure to fall slowly after sterilization. A2.1.4

Digest Broth (modified from Hartley, 1922, and Pope & Smith, 1932) Meat, finely minced 600 g Na2CO3 anhydrous 8g Water 1000 ml Pancreatic extract, Cole & Onslow's 20 ml

CULTURE MEDIA

CHCI3

20 ml

HC1, cone. 16 ml Add the alkali and the meat to the water, heat to 80°C, stir well and cool. Heat the infused mixture to 45-50 °C, add thepancreatic extract (A2.1.5) and chloroform, and maintain at 45-50 °C for 4-6 h with frequent stirring. Follow the course of digestion with the biuret test (Section A3.2). Add the acid, boil for 30 min and filter. Adjust to pH 8, boil for 30 min and filter. Adjust to pH 7.6, determine the amino acid nitrogen content (Section A3.3.2) and dilute the broth to contain 700-750 mg amino acid N2 per litre. Sterilize at 115 °Cfor 20 min. Notes. Cole & Onslow's pancreatic extract may be replaced by a commercial trypsin extract or powder. Pope & Smith recommend fractional addition of the enzyme during the digestion process. Digest Agar is Digest Broth gelled by the addition of 2% agar. A2.1.5 Pancreatic Extract (Cole & Onslow, 1916) Fresh pig pancreas 250 g Distilled water 750 ml Industrial methylated spirit 250 ml Add the water and spirit to the fat-free and minced pancreas in a 2 1 flask and stopper tightly. Shake thoroughly and leave at room temperature for 3 days with frequent shaking. Filter, add 0.1% cone. HC1 to the filtrate and mix well. Store at 4 °C. The cloudy precipitate which settlesin a few days may be filtered off. The extract may be expected to retain its activity for several months when stored at 4 °C. Note. A satisfactory extract should have a tryptic content of at least 50 units per ml (Section A3.1.8). A2.1.6 Infusion Broth (modified from Wright, 1933) Meat, minced 450 g Water 1000 ml Peptone 10 g NaCl 5g Allow the meat to infuse in the water overnight at 4 °C. Skim the fat from the infused mixture, add the peptone and salt and boil for 30 min. Filter, adjust to pH 8.4 and boil for 20 min. Filter, adjust to pH 7.6, and sterilize at 115 °C for 20 min. Infusion agar is Infusion Broth gelled by the addition of 2% agar.

A2.1.7 CYLG Broth (Marshall & Kelsey, 1960) Casein digest 10 g Marmite (yeast extract) 5g Sodium glycerophosphate 10 g Potassium lactate, 50% aq. soln 10 ml Glucose 2g Inorganic salts soln 5 ml Distilled water 1000 ml Dissolve the ingredients by heating, mix, filter, and sterilize at 115 °Cfor 20 min. Inorganic salts solution: 10N-H2SO4

0.1ml

MgSO4-7H2O 4g MnSO 4 4H 2 O 0.4 g FeSO4-7H2O 0.4 g Water 100 ml Add the acid to the water and dissolve the salts without heating. Note. This basal medium may be supplemented by the addition of blood or serum, gelled by the addition of agar, and can form the basis of many standard media. It is reproducible, does not require pH adjustment, and can be prepared and stored in concentrated form. A2.1.8

Thiogh collate Broth (modified from Brewer, 1940) Peptone 15 g Yeast extract 5 g NaCl 5g Agar 1 g Thioglycollic acid 1 g Glucose 5g Methylene blue, 1% aq. soln 0.2 ml Water 1000 ml Dissolve the solids in the water with gentle heat. Add the thioglycollic acid and adjust to pH 8.5 with N-NaOH. Autoclave at 115 °C for 10 min. Toprevent darkening of the medium, screw-caps should be loosened during autoclaving. Adjust to pH 7.2, add the glucose and dye solution, mix well and sterilize at 115 °C for10 min. Notes. This medium should be stored in screw-capped containers in the dark at 4 °C(Cook & Steel, \959b). If more than 20% of the medium shows a green colour before use, it should be heated in a boiling water bath or steamer for 5-10 min and allowed to cool undisturbed; this treatment must not be repeated. Media containing thioglycollate tend to become inhibitory to anaerobes with prolonged storage.

* Beecham Bovril Brands, P.O. Box 18, Burton-on-Trent, DE14 2AB, UK.

193

APPENDIX A A2.1.9 Dorset Egg medium Egg yolk and white 800 ml (about 16 hen eggs) NaCl, 0.9% sterile aq. soln 200 ml Wash the eggs in 70% ethanol and lay on a sterile surface. Break the shells with a sterile knife and let the contents fall into a sterile flask; add the saline aseptically. Shake thoroughly to break up the yolks and produce a homogeneous mixture. Distribute 2 ml volumes into sterile 5 ml screwcapped (bijoux) bottles or 5 ml volumes into sterile 30 ml screw-capped (1 oz Universal or McCartney) bottles. Slope the containers in an inspissator and heat slowly to 75 °C. Maintain at this temperature for 1 h and repeat the process on each of the following two days. Note. As an alternative to inspissation for egg and serum media, several workers have described methods of autoclaving which give uniform slopes free from air bubbles. The following procedure may be used with manually operated autoclaves : place the tubes in the autoclave in an inclined position and close the door and all valves of the autoclave; allow steam to enter the chamber rapidly until the pressure reaches 1.1 kgf/cm2^. Maintain this pressure for 10 min; as the air has not been expelled, the temperature will rise slowly, preventing the formation of air bubbles during coagulation. After 10 min, open the air exhaust valve very slowly ensuring that a pressure of 1.1 kgf/cm2 is maintained until the valve is fully open; rapid pressure changes will cause disruption of the slopes. Maintain the steam pressure for 15 min and then close both the steam supply and air exhaust valves. Allow the autoclave to cool slowly and do not open it until at least 5 min after the pressure has fallen to atmospheric. A2.1.10 Nutrient Agar Nutrient Broth (A2.1.1) gelled by the addition of 2% agar. A2.1.11 Yeast Extract Agar (Report, 1956c, 1983) Yeast extract 3g Peptone 5g Agar 12 g Distilled water 1000 ml Dissolve the yeast extract and peptone in the water. Adjust the reaction to pH 7.3. Add the agar and steam to dissolve. Distribute in 15 ml amounts in tubes or universal containers and larger volumes in screw-capped bottles. Autoclave at 115 °Cfor 10 minutes. Like Yeastrel Agar (Zinnemann, 1960), this medium is free from X and V growth factors and can be used as a base for testing for them. t l 5 lbf/in2

194

A2.1.12 Mueller-Hinton Agar (Mueller & Hinton, 1941) Meat infusion 6g Casein hydrolysate 17.5 g Starch 1.5 g Agar 10 g Water 1000 ml Bring to the boil to dissolve the ingredients completely. Adjust pH, if necessary, to 7.4. Sterilize at 121 °C for 15 min. This medium, which was originally designed for Neisseria, contains starch to adsorb toxic substances produced during growth. A2.1.13 Saline Agar NaCl 8.5 g Agar 20 g Water 1000 ml Dissolve by steaming; sterilize at 115 °C for 20 min. A2.1.14 Water Agar Agar 20 g Water 1000 ml Dissolve by steaming; sterilize at 115 °C for 20 min. A2.2

Enriched and enrichment media

Basic culture media may be made more suitable for the growth of many organisms, especially if fastidious or slowgrowing, by the addition of nutritious substances such as blood, serum or sugars. Such enriched media are essentially non-selective. Substances may also be added to media in order to encourage the growth of particular organisms or groups of organisms and isolation procedures often use such enrichment media. Their distinction from selective media, which inhibit the growth of unwanted organisms, is often blurred so that several of the media in Section A3 could also be described and used as enrichment media. Many such media have been developed for these purposes. Some have but a brief trial and are then discarded; those that are successful may be further developed and produced commercially. It should be noted that Salmonella choleraesuis is inhibited by selenite media commonly used for enrichment and isolation of enteric bacteria. A2.2.1 Blood Agar Defibrinated blood 50 ml Nutrient Agar 950 ml Melt the Nutrient Agar, cool to 50 °C and add the blood aseptically. Mix and distribute in tubes or plates. Notes. Haemolysis can be seen better in layered Blood Agar plates. For such plates, a layer of Peptone Water Agar

CULTURE MEDIA is poured into the Petri dish, allowed to set, and the bloodcontaining medium is poured on top. Alternatively, a simple agar solution without nutrients may be used as the lower layer; it must however be made isotonic to prevent haemolysis of the blood in the upper layer. The inclusion of glucose in the basal medium for Blood Agar is not recommended as its presence inhibits haemolysin production by streptcocci (Fuller & Maxted, 1939). Blood Broth is prepared by the aseptic addition of 5% sterile defibrinated blood to the appropriate volume of Nutrient Broth. A2.2.2 Chocolate Agar Place a Blood Agar plate (medium down) in an incubator set at 65 °C for 1-1xli h until the medium assumes a uniform 'chocolate' colour. Alternatively, melt Nutrient Agar and cool to 50 °C. Add 5% sterile blood, mix and heat in a water bath to 80 °C with frequent mixing. Maintain at 80 °C until the medium has a 'chocolate' colour and distribute aseptically. A2.2.3 Fildes' peptic digest of blood (Fildes, 1920) Defibrinated sheep blood 25 ml NaCl, 0.9% sterile aq. soln 75 ml HC1, cone. 3 ml Pepsin 0.5 g Mix the blood and saline in a glass-stoppered bottle and add the pepsin and acid. Shake thoroughly and place in a water bath at 55 °C for 4 h with occasional shaking. Add 6 ml 5 N-NaOH and check that the pH is 7.0. Add 0.25 ml CHC13 as a preservative and shake thoroughly. The digest will keep for at least 12 months but should be stored at 4 °C. It is better to leave it too acid rather than too alkaline. Note. Before use the chloroform must be removed by heating gently in a water bath. A2.2.4 Fildes' Digest Agar Fildes' peptic digest of blood 50 ml Nutrient Agar 950 ml Maintain the Fildes' digest at 55 °C for 30 min to volatilize the chloroform. Then aseptically add it to the Nutrient Agar, previously melted and cooled to 55 °C. Mix and distribute aseptically. A2.2.5 Serum Agar Sterile serum 50 ml Nutrient Agar 950 ml Melt the Nutrient Agar, cool to 50-55 °C and aseptically add the serum. Mix and distribute. Serum Broth is prepared by the aseptic addition of 5% sterile serum to Nutrient Broth.

A2.2.6 Serum Glucose Agar (Jones & Morgan, 1958) Peptone 10 g Meat extract 5g NaCl 5g Agar 20 g Water 1000 ml Prepare this base as for Nutrient Agar (A2.1.10) and sterilize at 115 °C for 20 min.. Sterile horse serum 50 ml Glucose, 20% aq. soln 50 ml Nutrient base 1000 ml Add the serum and sterile glucose solution to the base, previously melted and cooled to 50-55 °C. Mix and distribute aseptically. A2.2.7 Glucose Broth Glucose, 20% aq. soln 50 ml Nutrient Broth 950 ml Sterilize the glucose solution by filtration and add aseptically to the Nutrient Broth. Mix and distribute aseptically. A2.2.8 Glycerol Agar Glycerol 50 ml Nutrient Agar 1000 ml Melt the Nutrient Agar, add the glycerol, mix and sterilize at 115 °C for 20 min. Note. Gordon & Smith (1953) used 7% glycerol in Soil Extract Agar (A2.6.35). A2.2.9

Tomato Juice Agar (modified from Kulp & White, 1932) Tomatoes 250 g Distilled water 500 ml Cut up the tomatoes and steam in the water for an hour or until they are pulped. Clarify through gauze and filter through paper. Peptone 10 g Peptonized milk 10 g Agar 20 g Tomato juice 400 ml Distilled water 600 ml Dissolve the solids in the water by heating. Add the tomato juice, mix, and sterilize at 115 °C for 20 min. The final pH value of the medium should be 6.0-6.2. A2.3

Differential and selective media

A2.3.1 Acetate Agar (Whittenbury, 1965a) Meat extract (powder) Peptone Yeast extract (powder) Tween 80

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5g 5g 5g 0.5 ml

APPENDIX A Glucose 10 g Agar 15 g Distilled water 850 ml Dissolve the ingredients by heating, adjust to pH 5.4 with 2 M-acetic acid and sterilize at 115 °C for 20 min. Before dispensing, add 100 ml of 2 M-acetic acid with sodium acetate buffer (pH 5.4) to the molten medium. Bottle and sterilize at 115 °C for 20 min. Note. Prepare the acetate buffer by adjusting a 2 M-solution of sodium acetate to pH 5.4 with 2 M-acetic acid. A2.3.2 Bile Agar Ox bile, dehydrated 10 g Nutrient Agar 1000 ml Melt the Nutrient Agar, add the bile (equivalent to 10% bile), mix and dissolve. Sterilize at 115 °C for 20 min. Cool to about 55 °C and distribute. For fastidious organisms, serum (5%) may be added after cooling to 55 °C. Notes. For 40% Bile Agar, use 40 g dehydrated ox bile per litre. Bile Agar can be used as whole plates, or as ditches in plates of Blood or Serum Agar. A2.3.3 Blood-Tellurite Agar 16 ml K2TeO3, 2% aq. soln Sterile blood 50 ml Infusion Agar 1000 ml Melt the Infusion Agar medium, cool to 50 °C and aseptically add the blood and sterile tellurite solution. The medium must not be heated after addition of the tellurite. Mix and distribute. Notes. Sterilize the tellurite solution by filtration, not by heat. The final concentration of tellurite in this medium is approximately 0.03% (1/3333). Several tellurite media have been described; Anderson et al. (1931) and Barksdale (1981) used a 'chocolate' (heated blood) agar base, and laked (lysed) blood was used by Wilson (1934) and Hoyle (1941). A2.3.4

Bordet-Gengou Agar (modified from Bordet & Gengou, 1906) Peptone 10 g NaCl 5g Glycerol 10 ml Soluble starch 2.5 g Agar 3g Water 1000 ml Make a smooth paste of the soluble starch with a few ml of the water. Dissolve the peptone, NaCl, and glycerol in the remaining water, heat, and add the starch suspension. Adjust to pH 7.5, add the agar and dissolve by heating. Sterilize this base at 115 °C for 20 min. For use, aseptically add 500 ml horse blood (warmed to

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45 °C) to 1000 ml of base melted and cooled to 55-60 °C. Mix and distribute. Notes. Medium in plates should be thick, at least 5 mm in depth, and must not be overdried; it should be bright red in colour (Bailey, 1933); a dark medium indicates old or overheated blood. Some brands of peptone are markedly inhibitory to the growth of Bordetella pertussis and the original formula of Bordet & Gengou did not include peptone. Dawson et al. (1951) consider that 30% blood is the minimum for a satisfactory product; however, haemolysis is not usually seen when the blood concentration exceeds 30%; the original medium contained 50%. Bacteriological charcoal can be used instead of or as well as blood (Ensminger et al, 1953; Brumfitt, 1959) and antibiotics may be added to inhibit the competing flora (Lacey, 1954; Regan & Lowe, 1977). A2.3.5

Bromthymol Blue Salt Teepol Agar (BTBST) (Kobayashi et al, 1963; Sakazaki, 1965/?) Meat extract 3.0 g Tryptone 10.0 g Sucrose 10.0 g Teepol 610 2.0 ml NaCl 30.0 g Bromthymol blue 0.08 g Agar 15.0 g Distilled water 1000 ml Dissolve by steaming; adjust pH to 7.8 and pour as plates for immediate use. Sterilize stock medium at 121 °C for 15 min. For use, melt by steaming. Note. Plates should not be overdried. A2.3.6

Buffered Charcoal Yeast Extract Agar (BCYE) with a-ketoglutarate for Legionella (Edelsteinefa/., 1981) N-(2-acetamido)-2-aminoethanesulphonic acid buffer (ACES) 10 g KOH 2.8 g Activated charcoal 1.5 g Yeast extract 10 g Agar 17 g a-ketoglutarate 1 g L-cysteine hydrochloride 4% (w/v) sterile aq. soln 10 ml Ferric pyrophosphate 28% (w/v) sterile aq. soln 10 ml Distilled water 1000 ml Add the ACES buffer to 900 ml of distilled water in a flask and warm to 50 °C until dissolved. Add the KOH pellets to the buffer. Mix together the activated charcoal, yeast extract, agar and a-ketoglutarate and add them to the flask,

CULTURE MEDIA rinsing with a further 80 ml of water. Sterilize at 121 °C for 15 min. Cool to 50 °C and add separately the filter-sterilized solutions of ferric pyrophosphate and L-cysteine hydrochloride. Adjust the pH if necessary to 6.9 ± 0.2 with N-HC1 or KOH. Cool to 45 °C and distribute aseptically. The plates will keep for 2 weeks at 5 °C in plastic bags. A2.3.7 Glucose Salt Teepol Broth (Akiyama et al, 1963) Meat extract 3g Tryptone 10 g NaCl 30 g Glucose 5g 0.002 g Methyl violet Teepol 610 4 ml Distilled water 1000 ml Dissolve in the cold and adjust the pH to 9.4. Sterilize at 121 °C for 15 min. Dispense aseptically as needed. A2.3.8

MacConkey Agar (modified from MacConkey, 1908; Report, 1956c, 1983) Peptone 20 g NaCl 5g Sodium taurocholate 5g Agar 20 g Lactose 10 g Neutral red, 1% aq. soln 10 ml Water 1000 ml Dissolve the peptone, NaCl and bile salt in the water by heating. Adjust to pH 8.0, boil for 20 min, cool and filter. Add and dissolve the agar by boiling and adjust to pH 7.4. Add the lactose and indicator solution, mix and sterilize at 115 °C for 20 min. Notes. The exact quantity of indicator depends on the depth of colour preferred. Sodium taurocholate, sodium tauroglycocholate or other satisfactory bile salt, including ox gall, may be used (see Section A3.1.5). The use of 0.1% Teepol 610 (an anionic detergent) in place of bile salt in MacConkey Agar was recommended by Jameson & Emberley (1956). For MacConkey Broth see Section A2.6.9. A2.3.9

Salt Colistin (Polymyxin) Broth (Sakazaki, 1972; Nakanishi & Murase, 1974) Yeast extract 3g Tryptone 10 g NaCl 20 g Colistin sulphomethate sodium (Colomycin) 0.5 x 106 i.u OR (Polymyxin B) (2.5 x 105 i.u.) Distilled water 1000 ml

Dissolve the yeast extract, tryptone and salt in the water. Adjust to pH 7-0. Autoclave at 121 °C for 15 min and allow to cool. Add the Colomycin or Polymyxin aseptically. Keep at 4 °C and dispense as required. A2.3.10 Thiosulphate-Citrate-Bile salt-Sucrose (TCBS) Agar (Akiyama et al, 1963; Sakazaki, \965b) Yeast extract 5g Peptone 10 g Sodium thiosulphate 10 g Sodium citrate 1 g Ox bile 8g Sucrose 20 g Sodium chloride 1 g Ferric citrate 1g Bromthymol blue 0.04 g Agar 15 g Distilled water 1000 ml Add all the ingredients to the water and bring to the boil to dissolve. Do NOT autoclave. Dispense and allow to cool. A2.4

Media for enhancing pigment production

A2.4.1 Potato slopes. Take several large potatoes and scrub them thoroughly under running water. Cut cylinders with an 18-20 mm cork borer, rejecting any that are bruised or diseased. Cut each cylinder obliquely into two and place each half in a 30 ml screw-capped bottle or 25 mm diameter tube with the thick end resting on a small plug of absorbent cotton. Fill the containers with water and steam for 30 min. Pour off the water and sterilize at 115 °C for 20 min. A2.4.2 Mannitol Yeast-Extract Agar Peptone 2.5 g NaCl 2.5 g Agar 20 g Water 1000 ml Dissolve the solids in the water by heating. Adjust to pH 8.0, boil for 30 min and filter. Mannitol 5g Yeast extract 2.5 g Adjust to pH 7.0, add the mannitol and yeast extract. Mix, dissolve, and sterilize at 115 °C for 20 min. A2.4.3

King's media - for pseudomonads (King, Ward &Raney, 1954) Medium A - for pyocyanin Peptone 20 g Glycerol 10 g 10 g K2SO4, anhyd. 1.4 g MgCl2, anhyd.

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APPENDIX A Agar 20 g Water 1000 ml Dissolve the constituents except agar by heating in the water. Adjust to pH 7.2 if necessary. Add the agar and dissolve and sterilize by autoclaving at 115 °C for 10 min. Medium B - for fluorescin Proteose peptone 20 g Glycerol 10 g 1.5 g K2HPO4 1.5 g MgSO4-7H2O Agar 20 g Water 1000 ml Proceed as for medium A above. A2.5

Media for carbohydrate studies

A2.5.1

Hugh and Leifson's OF medium (Hugh & Leifson, 1953) Peptone 2g NaCl 5g 0.3 g K2HPO4 Agar 3g Distilled water 1000 ml Bromthymol blue, 0.2% aq. soln 15 ml Dissolve the solids by heating in the water. Adjust to pH 7.1, filter, and add the indicator. Sterilize at 115 °C for 20 min. Add a sterile solution of glucose or other appropriate carbohydrate aseptically to give a final concentration of 1%. Mix and distribute aseptically in 10 ml volumes into sterile tubes of not more than 16 mm diameter.

A2.5.2 Peptone Water sugars. The method of preparation will depend on the indicator: Andrade's indicator. Adjust the reaction of 900 ml Peptone Water to pH 7.1-7.3 so that the addition of 10 ml of Andrade's indicator will bring it to pH 7.5. Sterilize at 115 °C for 20 min; this medium is pink when hot but the colour fades on cooling. Dissolve 5-10 g of the appropriate sugar in 90 ml of water and steam for 30 min or sterilize by filtration. Aseptically add this to the sterile Peptone Water with indicator, distribute into sterile test tubes with inverted inner (Durham) tubes and steam for 30 min. Other indicators. To 900 ml Peptone Water add 10 ml indicator solution (Bromcresol purple, bromthymol blue, or phenol red) and sterilize at 115 °C for 20 min. Dissolve 5-10 g of the appropriate sugar in 90 ml water and steam for 30 min or sterilize by filtration. Add this to the sterile Peptone Water with indicator, distribute into sterile tubes with inverted inner (Durham) tubes and steam for 30 min. Notes. The addition of some carbohydrates may cause an acid reaction; in these instances add sufficient 0.1 N-NaOH to restore the original colour.

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Where the solubility of a carbohydrate is low (Table A2), the required amount of solid material may be added to the base and, when dissolved, the complete medium is sterilized. A2.5.3 Broth sugars Broth-based sugars are used extensively in the USA and are recommended by the international subcommittee on Enterobacteriaceae (Report, 1958). They are also recommended for haemophili and streptococci. Peptone 10 g Meat extract 3g NaCl 5g Distilled water 1000 ml Andrade's indicator OR Phenol red aq. soln 10 ml Dissolve the solids in the water, add the indicator and adjust to pH 7.1-7.2. Sterilize at 115 °C for 20 min. Aseptically add 0.5-1% of the appropriate carbohydrate (sterilized by membrane filtration), mix, distribute into sterile tubes containing inverted inner (Durham) tubes, and steam for 30 min. Note. A small amount of agar (e.g. 0.1%) may be included. A2.5.4 Serum Water sugars Peptone 4g Na2HPO4 0.8 g 800 ml Distilled water 200 ml Sterile serum 10 ml Bromcresol purple, 0.2% aq. soln Dissolve the peptone and phosphate in the water, steam at 100 °C for 15 min and filter. Add the serum and steam for a further 15 min. Adjust pH to 7.5-7.8 and add the indicator. Sterilize at 115 °C for 10 min. Aseptically add 0.5-1% of the appropriate sugar as a sterile solution and distribute into sterile tubes. Note. It is an advantage to have perfectly clear media. Different batches of serum differ in their coagulability by heat, and occasionally the medium is cloudy when serum in the amount given above is used. It is a good plan to add varying amounts of each batch of serum (e.g. in concentrations of 10-25%) to tubes of the basal medium, to autoclave them, and to choose the highest concentration that does not show marked cloudiness after sterilization. A2.5.5

Ammonium Salt Sugars (ASS)(Smith, Gordon & Clark, 1952) (NH4)2HPO4 1g KC1 0.2 g MgSO4-7H2O 0.2 g Yeast extract 0.2 g

CULTURE MEDIA Agar 20 g Distilled water 1000 ml 4 ml Bromcresol purple, 0.2% aq. soln 0.04 g OR Bromythmol blue Add the solids to the water and dissolve by steaming. Add the indicator and sterilize at 115 °C for 20 min. Allow the basal medium to cool to about 60 °C and add the appropriate carbohydrate as a sterile solution to give a final concentration of 0.5-1%. Mix and distribute aseptically into sterile tubes which are inclined so that the medium sets as slopes. A2.5.6

Sugars for neisserias (Thompson & Knudsen, 1958) Digest Broth 1000 ml Agar 3g Phenol red, 0.2% soln 10 ml Sterile rabbit serum 50 ml Sugar, sterile 10% soln 100 ml Dissolve the agar in the broth by heating, add the indicator solution and sterilize at 115 °C for 20 min. Cool to about 55 °C and aseptically add the serum and sugar solution. Distribute into sterile tubes. Notes. The four sugars used to differentiate Neisseria species are glucose, lactose, maltose, and sucrose. Wilkinson (1962) recommends a solid medium containing hydrocele fluid for detecting acid production by Neisseria species. A2.5.7

Sugars for lactobacilli (de Man, Rogosa & Sharpe, 1960) MRS broth, modified 1000 ml Carbohydrate 20 g Chlorphenol red, 0.2% soln 20 ml Prepare the basal MRS broth as in Section A2.6.25, omitting the meat extract and glucose; adjust to pH 6.2-6.5, add the indicator, and sterilize at 115 °C for 20 min. Add the carbohydrate (mannose, xylose, and maltose should be filter-sterilized and added aseptically), mix, and distribute; steam for 30 min. A2.5.8 Nutrient sugar media with agar (1) For utilization tests Lactose (or Glucose) 10% Agar Peptone 5g Meat extract 3g Lactose (or glucose) 100 g Agar 20 g Distilled water 1000 ml Bromcresol purple, 0.2% soln 10 ml Dissolve the solids in the water by heating, adjust to pH 6.8 with 10 N-NaOH, filter, and add the indicator solution.

Sterilize at 115 °C for 20 min and distribute as plates or slopes. (2) Media for 3-ketolactose production from lactose Medium 1 Yeast extract 10.0 g Glucose 20.0 g Calcium carbonate 20.0 g Agar 11.0 g Distilled water 1000 ml Mix ingredients well to produce an even suspension. Adjust pH to 7.0-7.2 and sterilize at 115 °C for 20 min. Distribute in plates. Medium 2 Yeast extract 1.0 g Lactose 10.0 g Agar 11.0 g Distilled water 1000 ml Dissolve ingredients, adjust to pH 7.0-7.2 and sterilize at 115 °C for 20 min. Distribute in plates. A2.5.9 MacConkey Broth (Report, 1956c, 1983) Peptone 20 g NaCl 5g Sodium taurocholate 5g Water 1000 ml Bromcresol purple, 0.2% soln 5 ml Lactose 20 g Dissolve the peptone, NaCl and bile salt in the water by heating. Adjust to pH 8.0 and boil for 20 min. Cool, filter, and adjust to pH 7.4. Add the lactose and indicator solution, mix and distribute into tubes containing inverted inner (Durham) tubes. Sterilize at 115 °C for 15 min. *Note. See MacConkey Agar (Section A2.3.8) for remarks on bile salts. A2.5.10 MR-VP test medium Glucose-phosphate medium Peptone 5g K2HPO4 5g Distilled water 1000 ml Steam until the solids are dissolved, filter, and adjust to pH 7.5. Glucose 5g Add the glucose, mix and distribute 1.5 ml volumes into tubes. Sterilize at 115 °C for 10 min. Notes. For sterilization, the tubes must be placed in a solid-bottomed container to protect them from contact with steam; if this is neglected, the medium becomes straw-yellow in colour. Workers in the USA generally use 7 g peptone.

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APPENDIX A This medium is also used for the VP test; other media for the VP test are shown below. A2.5.11 VP test media Glucose-peptone medium (Abd-el-Malek & Gibson, 19486) Peptone 10 g Glucose 5g Distilled water 1000 ml Mix and dissolve by gentle heating. Filter, adjust to pH 7.6, and distribute into tubes. Sterilize at 115 °C for 10 min in a solid-bottomed container. Glucose-salt medium (Smith, Gordon & Clark, 1946) Proteose peptone 7g NaCl 5g Glucose 5g Distilled water 1000 ml Dissolve the solids in the water, distribute into tubes, and sterilize at 115 °C for 20 min in a solid-bottomed container. Semi-solid VP medium (Furniss, Lee & Donovan, 1978) Tryptone 10 g Yeast extract 5g NaCl 5g K2HPO4 5g Glucose 5g Agar 3g Dissolve ingredients by heating. Dispense in 2.5 ml volumes in bijoux bottles and sterilize at 115 °C for 10 min. A2.5.12 Media for dextran and levan production Sucrose Agar Sterile serum 50 ml Sucrose 50 g Digest agar 1000 ml Melt the Digest Agar, add the sucrose, and steam for 30 min. Cool to 55 °C, aseptically add the serum, and distribute into Petri dishes. Sucrose Broth Sucrose 50 g Infusion Broth 1000 ml Dissolve the sucrose in the broth, distribute in tubes and steam for 1 h. A2.5.13 Starch Agar Potato starch 10 g Distilled water 50 ml Nutrient agar 1000 ml Triturate the starch with the water to a smooth cream, and add to the molten Nutrient Agar. Mix, and sterilize at 115 °C for 10 min. Distribute into Petri dishes. Notes. This medium should not be filtered after adding the starch suspension. Overheating may hydrolyse the starch.

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A2.6

Miscellaneous media

A2.6.1 Aesculin Broth Aesculin 1 g Ferric citrate 0.5 g Peptone Water 1000 ml Dissolve the aesculin and iron salt in the Peptone Water, and sterilize at 115 °C for 10 min. Aesculin Agar is Aesculin Broth gelled by the addition of 2% agar. A2.6.2

Aesculin-bile Agar (Williams & Hirch, 1950; Swan, 1954) Ox bile, dehydrated 40 g Aesculin 1 g Ferric citrate 0.5 g Agar 15 g Nutrient Broth 1000 ml Dissolve all the ingredients except aesculin by heating. Allow to cool and then add the aesculin. Dispense in screwcapped bottles, sterilize at 115 °C for 20 min and allow to set as slopes. A2.6.3

Aesculin Blood Agar (Phillips, 1976) for anaerobic work Prepare an aqueous solution of aesculin (2% w/v) and ferric citrate (1% w/v) and steam at 100 °C for 5 minutes. Since aesculin at this concentration is completely soluble only when the solution is warm, spread pre-dried Blood Agar plate with 1.0 ml of the aesculin-ferric citrate solution at about 50 °C; this gives a final concentration of about 0.1% aesculin in the medium. Spot-inoculate plates and incubate anaerobically for 48 hours. Aesculin hydrolysis is indicated by blackening of the medium under and around bacterial growth. A2.6.4 Arginine media Arginine Broth (Niven, Smiley & Sherman, 1942) Peptone (tryptone) 5g Yeast extract 5g K2HPO4 2g Glucose 0.5 g Arginine monohydrochloride 3g Distilled water 1000 ml Dissolve by heating, adjust to pH 7.0, boil, filter, and sterilize at 115 °C for 20 min. Note. L-arginine monohydrochloride is dextro-rotatory and in consequence was incorrectly called D-arginine monohydrochloride in the original paper of Niven et al. Arginine Agar (Thornley, 1960) Peptone 1.0 g NaCl 5.0 g

CULTURE MEDIA K2HPO4 0.3 g 1.0 ml Phenol red, 1.0% aq. soln 10.0 g L(+)arginine hydrochloride 3.0 g Agar 1000 ml Distilled water istribute Dissolve the solids in the water, adjust to pH 7.2, distribute into tubes or screw-capped (6 mm) bottles to a depth of about 16 mm (3.5 ml) and sterilize at 121 °C for 15 min. A2.6.5

Bicarbonate Agar with Serum (Burdon, 1956; Burdon & Wende, 1960) for encapsulation of B. anthracis Nutrient Broth (without added salt) 100 ml Yeast extract 0.3 g Glucose 0.5 g Agar 2.5 g Make up with gentle heating, in distilled water. Sterilize by autoclaving at 115 °C for 10 minutes. Add 7% solution of NaHCO3, sterilized by filtration, to the sterile molten agar medium to give a final concentration of 0.7% bicarbonate. Then add sterile bovine serum albumin, fraction V, to give a final concentration of 0.7% (w/v); alternatively, in order of preference, add human, sheep, calf or horse serum to a final concentration of 20% (v/v). Pour into plates. A2.6.6

Casein Agar (Milk Agar) (modified from Hastings, 1903) Milk, skim 500 ml Nutrient Agar, double-strength 500 ml Prepare the skim-milk as in Section A2.6.23 and sterilize by heating at 115 °C for 10 min. Cool to about 50 °C and add to the double-strength Nutrient Agar (Section A2.1.1) melted and cooled to 50-55 °C. Mix and distribute in Petri dishes or tubes. Note. As the acid produced by lactose fermentation had an inhibitory effect on the hydrolysis of casein, Eddy (1960) recommended dialysis of milk before adding to the basal medium. Alternatively, a 10% aqueous solution of skimmed milk powder may be used. This is distributed in screw-capped bottles and autoclaved at 121 °C for 5 min. For use, add 50 ml of the skimmed milk solution to 100 ml of a melted sterile Nutrient Agar solution (2.5% in distilled water), mix and pour into Petri dishes. A2.6.7 Citrate media Christensen's medium (Christensen, 1949) Sodium citrate Glucose Yeast extract L-cysteine hydrochloride Ferric ammonium citrate

3g 0.2 g 0.5 g 0.1 g 0.4 g

KH2PO4 lg NaCl 0.08 g Na2S2O3 20 g Agar 6 ml Phenol red, 0.2% aq. soln 1000 ml Distilled water Dissolve the solids in the water by heating and filter; adjust to pH 6.8-6.9, add the indicator, and sterilize at 115 °C for 20 min. Note. This medium is also suitable for demonstrating H2S production; if it is not to be used for this purpose the Na2S2O3 and ferric ammonium citrate can be omitted. Koser's medium (modified from Koser, 1923; Report, 1983) NaCl 5g MgSO4-7H2O 0.2 g 1g NH4H2PO4 1g K2HPO4 Citric acid 2g Distilled water 1000 ml Dissolve the salts in the water. Add the citric acid to the salts solution and adjust to pH 6.8 with N-NaOH. Dispense in 5 ml volumes in screw-capped containers and autoclave at 115 °C for 20 min. The medium should be colourless. All glassware must be chemically clean and alkali-free (see Section A 1.1). Note. In his original paper, Koser (1923) used 2 g sodium citrate or 2.77 g of the hydrated salt in place of the citric acid in the formula given above; it is not known which of the three sodium salts of citric acid was used although Koser stated that it had 5xk molecules of water of crystallization. Simmons' medium (modified from Simmons, 1926) This is the modified Koser's Citrate medium (above) incorporating 0.008% bromthymol blue (i.e. 20 ml of 0.4% aqueous solution per litre), and gelled by the addition of 2% agar. The colourless or faintly green medium is rendered blue by bacterial growth if citrate is utilized. A2.6.8

Organic acids as carbon sources (Gordon & Mihm, 1957) NaCl 1g 0.2 g MgSO4-7H2O 1g (NH4)2HPO4 0.5 g KH2PO4 Organic acid (sodium salt) 2g Phenol red, 0.2% aq. soln 4 ml Agar 20 g Distilled water 1000 ml Dissolve the agar by steaming in about 800 ml of the water. Dissolve the salts in the remainder of the water and add to

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APPENDIX A the agar solution. Adjust to pH 6.8 and add the indicator solution. Sterilize at 115 °C for 20 min. Notes. Organic acids used as their sodium salts include: acetate, benzoate, citrate, lactate, oxalate, propionate, pyruvate, succinate and tartrate; malate is used as its calcium salt, and mucate as the free acid. This medium resembles the modified Simmons' Citrate medium (above) but contains less NaCl and a different indicator. A2.6.9 Decarboxylase media M0ller's medium (M0ller, 1955) Peptone 5g Meat extract 5g Pyridoxal 5 mg Glucose 0.5 g Bromcresol purple 0.2% aq. soln 5 ml Cresol red, 0.2% aq. soln 2.5 ml Distilled water 1000 ml Dissolve the solids in the water by heating. Adjust to pH 6.0. Add the indicators, mix, and distribute into four equal volumes. Sterilize at 115 °C for 20 min. For use make the following additions and then re-adjust to pH 6.0 if necessary: 1. L-arginine hydrochloride 1% 2. L-lysine hydrochloride 1% 3. L-ornithine hydrochloride 1% 4. No addition Distribute the four media in 1-1.5 ml volumes into small rimless tubes (67 x 10 mm or 3 x 3/s in) containing sterile medicinal grade liquid paraffin to a height of about 5 mm above the medium. After distribution sterilize at 115 °C for 10 min or by steaming at 100 °C for 30 min on three successive days. Notes. M0ller specified Orthana Special peptone but other peptones are satisfactory. When DL-amino acids are used, the concentration should be 2%. If glutamic acid decarboxylase activity is also to be investigated, the basal medium should be divided into five portions, to the fifth of which 1% L-glutamic acid is added. Falkow's medium (modified from Falkow, 1958) Peptone 5 g Yeast extract 3 g Glucose 1g Bromcresol purple, 0.2% aq.soln 10 ml Distilled water 1000 ml Dissolve the solids in the water, adjust to pH 6.7, and add the indicator solution. Sterilize at 115 °C for 20 min. Divide into four equal volumes and make the following additions: 1. L-arginine hydrochloride 0.5% 2. L-lysine hydrochloride 0.5%

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3. L-ornithine hydrochloride 0.5% 4. No addition Re-adjust pH to 6.7 if necessary. Dispense in 2 ml volumes in small tubes and sterilize at 115 °C for 10 min. A2.6.10 DNA Toluidine-blue Agar media (Modified from Schreier, 1969) for DNase Method 2. Tryptose 20 g Deoxyribonucleic acid 2g NaCl 5g Toluidine blue 0.1 g Agar 20 g Distilled water 1000 ml Dissolve constituents in the distilled water, autoclave at 121 °C for 15 min. and pour into Petri dishes. (Lior & Patel, 1987) for DNase Method 3 Deoxyribonucleic acid 0.3 g 0.01 M-calcium chloride 1 m Sodium chloride 10 g Agar 6.5 g 0.05 M-Tris buffer pH 9.0 1000 ml Add all the ingredients to the buffer solution and heat to boiling until the DNA and agar are completely dissolved. Cool to 50 °C, add 2.5 ml of 3% toluidine blue '0' and mix well. Does not require sterilization. Store in the dark. Note. Tris buffer requires a pH electrode with calomel reference. Tris buffers are available commercially which do not require adjustment. A2.6.11 Ferric Bisulphite Pyruvate (FBP) medium for rapid H2S test (method 7) Nutrient Broth 100 ml 1.18 g Na2HPO4 (anhydrous) 0.23 g KH2PO4 (anhydrous) Agar 2g Water 900 ml Dissolve the ingredients in the water by heating, sterilize at 121 °C for 15 min and cool to 48 °C. Add the following ingredients in the manner described below :FeSO4-7H20, 10% soln 10 ml Sodium metabisulphite, 10% aq. soln 10 ml Sodium pyruvate, 10% aq. soln 10 ml Add the ferrous sulphate solution to the metabisulphite solution, mix well, then add to the pyruvate solution, mix again and add to the basal medium. Adjust to pH 7.3 with 1 N-NaOH. Store at 4 °C and prepare fresh every few weeks. A2.6.12 Ferrous chloride Gelatin medium (Report, 1958) for H2S test (Method 2) Meat extract 7.5 g Peptone 25 g

CULTURE MEDIA NaCl 5g Gelatin 120 g Distilled water 1000 ml 5 ml FeCl2, 10% aq. soln Prepare the base as for Nutrient Gelatin (A2.6.14). After sterilization and before the medium gels, add the freshly prepared FeCl2 solution. Dispense into narrow tubes, cool immediately, and seal the tubes with corks that have been soaked in hot paraffin wax. A2.6.13 Fluorescence-denitrification medium (FN medium of Pickett & Pedersen, 1968) Proteose peptone 10.0 g MgSO4-7H2O 1.5 g K2HPO4 1.5 g KNO3 2.0 g NaNO2 0.5 g Agar 15.0 g Water 1000 ml Dissolve by heating. Adjust pH to 7.2; dispense in 4 ml volumes in 12 mm tubes or about 10 ml in 28 ml (Universal or McCartney) screw-capped bottles. Sterilize at 121 °C for 10 min. Set at an angle to give a slope and butt of approximately equal length. For fluorescence, inoculate the surface; for denitrification, stab-inoculate the butt. A2.6.14 Gelatin media Gelatin Agar (Frazier, 1926) Gelatin 4g Distilled water 50 ml Nutrient Agar 1000 ml Soak the gelatin in the water and, when thoroughly softened, add to the melted Nutrient Agar. Mix, and sterilize at 115 °C for 10 min. Distribute into plates. Note. After growth, gelatinase activity is demonstrated by treating with a solution of 15% mercuric chloride in 1 N-HCI. Zones of clearing develop around gelatinolytic colonies. For safety reaons, 30% trichloroacetic acid is preferable to use of the acid mercuric choloride solution. Nutrient Gelatin Meat extract 3g Peptone 5g Gelatin 120 g Water 100 ml Add the gelatin to the water and allow to stand for 15-30 min. Heat to dissolve the gelatin; add and dissolve the other constituents. Adjust to pH 7.0, and sterilize by heating at 115 °C for 20 min. Note. This medium must not be overheated.

A2.6.15 Gluconate Broth (Shaw & Clarke, 1955; Carpenter, 1961) Peptone 1.5 g Yeast extract 1 g K2HPO4 1 g Potassium gluconate 40 g Distilled water 1000 ml Dissolve in the water by heating. Adjust to pH 7.0. Filter, and sterilize at 115 °C for 20 min. Note. The potassium gluconate may be replaced by 37.25 g of sodium gluconate. A2.6.16 Glucose Phenolphthalein Broth (Clarke, 19536) Glycine buffer Glycine 0.6 g NaCl 0.35 g Distilled water, freshly boiled 60 ml O.lN-NaOH 40 ml Dissolve the glycine and NaCl in the water and add the alkali. 1 % Glucose Broth (A2.2.7) 900 ml Glycine buffer 100 ml Phenolphthalein, 0.2% aq. soln 5 ml Mix and keep overnight at 4 °C in a stoppered flask. Sterilize by filtration and aseptically distribute into sterile 5 ml screw-capped bottles leaving as little air space as possible. Incubate overnight to check sterility and discard any bottles not showing a definite pink colour. Note. This medium should be used as soon as possible after preparation. A2.6.17 Hippurate media Hippurate Agar (modified from Hajna & Damon, 1934; Thirst, 1957a) NaCl 5g MgSO4-7H2O 0.2 g 1 g NH4H2PO4 1 g K2HPO4 Sodium hippurate 3g Agar 20 g Distilled water 1000 ml Phenol red, 0.2% soln 5 ml Dissolve the solids in the water by heating, adjust to pH 6.8-7.0, and add the indicator. Sterilize at 115 °C for 20 min. Hippurate Broth (Hare & Colebrook, 1934) Sodium hippurate 10 g Infusion Broth 1000 ml Dissolve the hippurate in the broth and sterilize at 115 °C for 20 min. Alternatively, for streptococci, add sodium hippurate

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APPENDIX A (10% solution sterilized b^ membrane filtration) to Nutrient Broth containing 0.1 % glucose to give a final concentration of 1 % hippurate. A2.6.18 KCN Broth (modified from M0ller, \954b; Rogers & Taylor, 1961) Peptone 3g NaCl 5g KH2PO4 0.225 g Na2HPO4-2H2O 5.64 g Distilled water 1000 ml Dissolve the solids in the water and distribute in 100 ml volumes in screw-capped containers. Sterilize at 115 °C for 20 min. For use, add 1.5 ml of a freshly prepared 0.5% KCN solution in sterile water to 100 ml of base. Mix and distribute aseptically in 1 ml volumes in sterile 5 ml screwcapped (bijoux) bottles. Note. Store at 4 °C and use within 4 weeks of preparation. A2.6.19 Loeffler serum slopes Glucose 5g Nutrient Broth 250 ml Serum, sterile 750 ml Dissolve the glucose in the Nutrient Broth and steam for 10 min. Cool and add aseptically to the filtered serum. Distribute 2.5 ml volumes into sterile tubes and heat at 75 °C for 2 h on each of 3 successive days, keeping the tubes in such a position that a slope is made. Notes. It is essential that during the first period of heating the temperature is raised slowly, otherwise the surface serum will be coagulated before the dissolved air has been driven off and the finished medium will have an uneven surface. For an alternative sterilization procedure see Section A2.1.9. A2.6.20 Lowenstein-Jensen medium (modified from Lowenstein, 1931; Jensen, 1932) Mineral Salt solution 2.4 g KH2SO4 0.2 g MgSO4-7H2O Glycerol 12.0 ml Distilled water 600 ml Dissolve the ingredients in the water and autoclave at 110 °Cfor 15 minutes. Malachite Green solution Malachite green 0.2 g Distilled water 10 ml Mix and incubate at 37 °C or place in water bath at 56-60 °C to dissolve. Autoclave at 115 °C for 10 min. or sterilize by membrane filtration.

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Egg Mixture Immerse twenty hens' eggs in 70% ethanol and leave for 15 min. Remove to a clean tray and allow to dry. Break into a sterile jar containing glass beads and shake well to mix the white and yolks. Strain through sterile gauze into a sterile beaker and add the mineral salt and malachite green solutions. Mix thoroughly and distribute in 5.0 ml volumes in 30 ml screw-capped (Universal or McCartney) bottles. Screw caps tightly, place the bottles almost horizontally in a hot-air oven and inspissate by a single heating; raise the temperature to 75 °C and hold it between 75 °C and 85 °C for 45 minutes. For characterization tests distribute in 2 ml volumes in 7 ml bottles and inspissate as above. Lowenstein-Jensen medium with TSC (Thiosemicarbazone; thiacetazone) 0.5% TSC in dimethylformamide 0.6 ml Lowenstein-Jensen Medium 300 ml Dissolve the TSC (thiosemicarbazone; thiacetazone) in the dimethylformamide solvent (0.6 ml), dilute with twice its volume of sterile distilled water and add to 300 ml of liquid LJ medium. Mix and dispense before inspissation. Final concentration of TSC = approximately 10 mg/ml. Lowenstein - Jensen medium with PNB (p-nitrobenzoic acid) Add 300 mg PNB (p-nitrobenzoic acid) to 10 ml sterile distilled water. Add 1 drop 0.4% aqueous phenol red solution and 2 ml of 1 N-NaOH. Stir or shake until dissolved. With continuous shaking, add 1 N-HC1 drop by drop until the indicator just turns yellow when the PNB will precipitate out. Back titrate with a small amount of 1 N-NaOH until the indicator just turns pink when the precipitate will redissolve. Add to 600 ml liquid LJ medium, mix and distribute before inspissation. Final concentration of PNB = approximately 500 mg/ml. A2.6.21 Lecithovitellin (LV) Agar (Macfarlane, Oakley & Anderson, 1941) Lecithovitellin solution (egg-yolk saline) Hen eggs 4 NaCl, 0.85% soln 1000 ml Separate the yolks from the whites and beat the yolks in the saline to form a homogeneous mixture. Add 25 g kieselguhr (diatomite), mix and clarify by filtration through paper. Sterilize the clarified material by filtration. Notes. If not for immediate use, distribute the solution aseptically into sterile containers and keep in a refrigerator; it may decrease in sensitivity slightly over a period of 2 weeks or so, but most batches remain unaltered in sensitivity for months; if a precipitate appears, the batch should be discarded. Yolks vary in size and in order to overcome the varia-

CULTURE MEDIA tions in egg-yolk saline due to this, McGaughey & Chu (1948) used 5% w/v egg-yolk. Billing & Luckhurst (1957) claim that filtration is easier when distilled water is used in place of saline. Lecithovitellin Agar Lecithovitellin solution 100 ml (or Egg Yok Emulsion) Nutrient Agar 900 ml Melt the Nutrient agar and cool to about 55°C. Add the lecithovitellin solution aseptically, mix and pour plates. A2.6.22 Malonate-phenylalanine medium (Shaw & Clarke, 1955) 2g (NH4)2SO4 0.6 g K2HPO4 KH2PO4 0.4 g NaCl 2g Sodium malonate 3g DL-phenylalanine 2g Yeast extract 1 g Distilled water 1000 ml Bromthymol blue, 0.2% aq. soln 12.5 ml Dissolve the solids in the water by heating. Filter, add the indicator solution, and sterilize at 115 °C for 20 min. Notes. When L-phenylalanine is used, only 1 g is needed. Another medium, Phenylalanine Agar, for detecting phenylalanine deaminase is described in Section A2.6.30. A2.6.23 Milk media Litmus Milk. Stand fresh whole milk in a refrigerator overnight and remove the milk below the cream layer by siphoning, taking care to avoid the cream layer. Steam for 1 h and cool in a refrigerator. Filter and measure the volume of filtrate. Add sufficient litmus solution (Section A 1.2.2) to give a bluish-purple colour and sterilize at 115 °C for 10 min or by steaming for 30 min on each of 3 successive days. After heating, this medium is colourless but the colour returns on cooling. Notes. Overheating must be avoided to prevent caramelization. Homogenized milk is unsuitable. Purple Milk, as above but replace litmus solution with 10 ml 0.2% aqueous bromcresol purple solution per 1000 ml skim-milk. Ulrich's Milk (Ulrich, 1944) as litmus milk but use chlorphenol red (0.0015%) as the pH indicator, and methylene blue (0.0005%) as a redox potential indicator. To 1000 ml skim-milk, add 7.5 ml 0.2% aqueous chlorphenol red solution and 2.5 ml 0.2% aqueous methylene blue solution. A2.6.24 Motility media Peptone

10 g

Meat extract 3g NaCl 5g Agar 4g Gelatin 80 g Distilled water 1000 ml Soak the gelatin in the water for 30 min, add the other ingredients, heat to dissolve, and sterilize at 115 °C for 20 min. Note. Hajna (1950) modified this formula to permit the simultaneous detection of H2S production by the addition of 0.2 g cysteine and 0.2 g ferrous ammonium sulphate; 2 g sodium citrate was added to clarify the medium and provide an extra nutrient for citrate-utilizing bacteria. Sulphide-indole-motility (SIM) medium Tryptone 30 g Meat extract 3g Na2S2O3-5H2O 0.5 g Cysteine hydrochloride 0.2 g NaCl 5g Agar 5g Distilled water 1000 ml Add the constituents to the water and heat to dissolve. Dispense into bottles or tubes and sterilize by autoclaving at 121 °C for 15 minutes. For use, stab-inoculate cultures and incubate for 18-24 hours at 37 °C. Diffusion of growth from the line of inoculum indicates motility; blackening, H2S production; and the development of a red colour within one hour after the addition of Kovacs' reagent (Section C1.7) indicates indole production. A2.6.25 MRS Lactobacillus Broth (de Man, Rogosa & Sharpe, 1960) Peptone 10 g Meat extract 10 g Yeast extract 5g Glucose 20 g Polyoxyethylene sorbitanmono-oleate (Tween 80) 1 ml 2g K2HPO4 5g CH3COONa.3H20 Triammonium citrate 2g MgSO44H2O 0.02 g 0.05 g MnSO4-4H2O Distilled water 1000 ml Dissolve the ingredients in the water, and sterilize at 115 °C for 20 min or at 121 °C for 15 min. Notes. This medium may be gelled by the addition of 2% agar. As a basal medium for carbohydrate studies this formula is modified by the omission of glucose and meat extract (Section A2.5.7).

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APPENDIX A A2.6.26 Nitrate media Nitrate-Blood Agar (Cook, 1950) KNO3, sterile 20% aq. soln 5 ml Horse blood 60 ml Nutrient Agar or Digest Agar 1000 ml Melt the Nutrient Agar, cool to 50-55 °C, add the sterile KNO3 solution and horse blood aseptically, mix, and distribute into Petri dishes. Nitrate Broth KNO3 1g Nutrient Broth 1000 ml Dissolve the KNO3 in the broth, distribute into tubes containing inverted inner (Durham) tubes, and sterilize at 115 o Cfor20min. A2.6.27 Nitrite Broth NaNO2 0.01 g Nutrient Broth 1000 ml Dissolve the nitrite in the broth, distribute into tubes and sterilize at 115 °C for 20 min. A2.6.28 ONPG Broth (Lowe, 1962) ONPG 6g 0.01 M-Na2HPO4 1000 ml Dissolve at room temperature the ONPG (O-nitro-phenylD-galactopyranoside) in the phosphate solution at pH 7.5; sterilize by filtration. Note. This solution should be stored in a refrigerator at 4 °C and protected from light. ONPG soln 250 ml Peptone Water 750 ml Aseptically add the ONPG solution to the Peptone Water (Section A2.1.2) and distribute in 2.5 ml volumes in sterile tubes. Notes. The medium is stable for a month when stored at 4°C. Sodium Potassium Magnesium (SPM) Broth may be used instead of Peptone Water. SPM broth contains: Tryptone, 10 g; NaCl, 10 g; KC1, 1 g; MgCl2-6H2O, 4 g; distilled water, 1000 ml. A2.6.29 Phenolphthalein Phosphate Agar Phenolphthalein diphosphate, Na salt 1 % aq. soln. Sterilize by filtration and store at 4 °C. Phenolphthalein phosphate soln 10 ml Nutrient Agar 1000 ml Melt the Nutrient Agar and cool to 45-50 °C. Add the phenolphthalein phosphate solution aseptically, mix, and distribute into Petri dishes.

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A2.6.30 Phenylalanine Agar (Ewing, Davis & Reavis, 1957) DL-phenylalanine 2g Yeast extract 3g Na2HPO4 1g NaCl 5g Agar 20 g Distilled water 1000 ml Dissolve the ingredients by heating in the water, filter, tube, and sterilize at 115 °C for 20 min. Solidify in a slanting position to give a long slope. Notes. When L-phenylalanine is used, only 1 g is required. A combined medium (with malonate) is described in Section A2.6.22. A2.6.31 Polyhydroxybutyrate (PHB) Agar (Owens & Keddie, 1968) Basal Medium K2HPO4 0.52 g KH2PO4 0.375 g (NH4)2SO4 0.5 g 0.05 g CaCl2 MgSO4-7H2O 0.2 g NaCl 0.1 g FeCl3-6H3O 0.01 g 0.002 g MnSO4-4H2O 0.002 g Na2MoO4-2H2O New Zealand Agar (Davis) 13.0 g Distilled water 1000 ml Bring to boil all ingredients except the agar, cool to room temperature and filter. Add the agar and boil to dissolve. Check reaction and adjust to pH 7.0. Dispense in 20 ml volumes in Universal bottles and sterilize by autoclaving at 115 °C for 15 min. Complete medium Melt 20 ml of mineral base by heating at 115 °C for 10 min. Allow to cool to 60 °C. Add 0.1 g of the sodium salt of DL-(3-hydroxy butyric acid to the molten base, mix gently and distribute into Petri dishes. Store at 4 °C. A2.6.32 Pyruvate Fermentation medium Tryptone Yeast extract Sodium pyruvate Bromthymol blue (ethanolic solution, 4 mg/ml) K2HPO4 Agar Distilled water

10 g 5g 10 g 10 ml 5g 10 g 1000 ml

CULTURE MEDIA Dissolve the solid ingredients by heat; allow to cool and add the indicator solution. Mix, adjust pH to 12-1A and dispense in small screw-capped bottles or tubes. Sterilize at 115 °C for 20 min. Allow to cool as slopes. A2.6.33 Salt Broth. Nutrient Broth (A2.1.1) with the NaCl content increased to 65 g/1000 ml, or the amount needed to give the concentration required. Note. For halophilic vibrio species the salt concentration of all test media should be 1.5-3%. A2.6.34 Selenite Broth (Hobbs & Allison, 1945, modified by Lapage & Bascomb, 1968) Peptone 5g Mannitol 4g Na2HPO4-12H2O 4.3 g NaH2PO4-2H2O 2.8 g Water 1000 ml Dissolve the ingredients in the water with gentle heat. Adjust pH to 7.2; sterilize at 121 °C for 30 min. Sodium hydrogen selenite, 40% aq. soln Sterilize by filtration and store at 4 °C. On the day of use add 1 ml 40% selenite solution aseptically to 99 ml of the broth; mix, and distribute 4 ml volumes into sterile 150 x 16 mm (6 x 5/s in) test tubes; use metal closure caps. A2.6.35 Soil-extract Agar (Gordon & Smith, 1953) Soil extract Garden soil, air-dried 1000 g Water 2400 ml Sift the air-dried soil through a fine (no.9) mesh sieve and add to the water. Mix well and heat the suspension in an autoclave at 121 °C for 1 h or at 126 °C for 20-30 min. Stir and filter through paper. Notes. If the soil is rich in organic matter, 500 g will suffice. If the soil is insufficiently dry the filtrate will be turbid, but may be clarified by the addition of talc, and then refiltered. Soil-extract Agar Peptone 5g Meat extract 3g Agar 20 g Soil extract 1000 ml Heat to dissolve, adjust to pH 7.0, and sterilize at 115 °C for 20 min. A2.6.36 Medium for preparation of Streptomyces extract for streptococcal grouping (modified fromMaxted, 1948) NaCl 5g 2g K2HPO4

MgSO4-7H2O 1 g CaCl2 0.04 g 0.02 g FeSO4-7H2O ZnSO4-7H2O 0.01 g Yeast extract 5g Agar 20 g Distilled water 1000 ml Dissolve the agar by steaming in 750 ml of the water. Dissolve the salts and yeast extract in the remaining water and add to the agar solution. Mix and sterilize at 115 °C for 20 min. Glucose, 20% sterile aq. soln 25 ml For use, melt and cool to about 60 °C, aseptically add sterile glucose solution, and distribute into sterile Roux bottles. A2.6.37 Todd-Hewitt Broth (modified from Todd & Hewitt, 1932) Meat, minced 450 g Water 1000 ml Soak the meat in the water overnight at 4 °C; skim the fat from the infused mixture, heat to 85 °C, and maintain at this temperature for 30 min; filter and adjust the volume if necessary to 1000 ml. Peptone 20 g 10N-NaOH 2.7 ml 2g NaHCO3 NaCl 2g Na2HPO4-12H2O 1 g Glucose 2g Add the peptone to the meat infusion; mix well and add the alkali, followed by the other ingredients; slowly heat to boiling-point and boil for 30 min; allow to cool, adjust to pH 7.8, filter and sterilize at 115 °C for 20 min. Note. When this medium is to be used for streptococcal type identification, it is essential that the peptone used does not encourage production of the proteinase that destroys the M antigen (Elliott, 1945). Suitable peptones for this medium include Difco Neopeptone and Evans' peptone. A2.6.38 Triple Sugar Iron Agar (TSI) (Report, 1958) Meat extract 3g Yeast extract 3g Peptone 20 g Glucose 1 g Lactose 10 g Sucrose 10 g FeSO4-7H2O 0.2 g OR [Ferric citrate] [0.3 g] NaCl 5g 0.3 g Na2S2O3-5H2O Agar 20 g

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APPENDIX A Distilled water 1000 ml Phenol red, 0.2% aq. soln 12 ml Heat to dissolve the solids in the water, add the indicator solution, mix and dispense into tubes. Sterilize at 115 °C for 20 min and cool to form slopes with deep butts about 3 cm long. A2.6.39 Tween 80 medium (Sierra, 1957) Peptone 10 g NaCl 5g 0.1 g CaCl2-2H2O Agar 20 g Distilled water 1000 ml Dissolve by steaming; adjust the pH to 7.4. Volumes of 500 ml are sterilized in flasks, which are cooled to 40-50 °C. Tween 80 is sterilized at 121 °C and 5 ml added aseptically to each flask to give a final concentration of 1%. Dispense into Petri dishes. A2.6.40 Tyrosine Agar (Gordon & Smith, 1955) Peptone 5g Meat extract 3g Agar 20 g Distilled water 100 ml L-tyrosine 5g Prepare the base as for Nutrient Agar (Section A2.1.1); add the tyrosine, mix well to produce a uniform suspension of the insoluble tyrosine, and sterilize at 115 °C for 20 minutes and allow to cool to 60 °C. Distribute into Petri dishes. A2.6.41 Urea media Christensen's medium (Christensen, 1946) Peptone Ig NaCl 5g KH2PO4 2g Agar 20 g Distilled water 1000 ml Dissolve the ingredients by heating, adjust to pH 6.8, filter, and sterilize at 115 °C for 20 min. Glucose 1 g Phenol red, 0.2% aq. soln 6 ml Add to the molten base, steam for 1 h, and cool to 50-55 °C. Urea, 20% aq. soln 100 ml Sterilize by filtration and add aseptically to the base cooled at 50-55 °C. Distribute the medium aseptically into sterile containers and allow to cool as slopes or plates. SSR medium (Stuart, van Stratum & Rustigian, 1945) KH2PO4 9.1 g Na2HPO4 9.5 g Yeast extract 0.1 g Urea 20 g

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Phenol red, 0.2% aq. soln 5 ml Distilled water 1000 ml Dissolve the solids in the water without heating, check and if necessary adjust the pH to 6.8, add the indicator, and sterilize by filtration. Aseptically distribute into sterile, chemically clean tubes. Alternatively, the base can be prepared and sterilized by autoclaving and the urea added aseptically as a sterile solution. Maslen's (1952) liquid modification of Christensen's medium Peptone 1 g Glucose 1 g Na2HPO4 1.2 g KH2PO4 0.8 g NaCl 5g Phenol red 0.2% aq. soln 5 ml Distilled water 1000 ml Dissolve the solids in the water. Check and if necessary adjust the pH to 6.8. Sterilize at 115 °C for 20 mins. Cool to 55 °C and aseptically add 50 ml sterile 40% urea solution. Mix well and distribute in 2.5 ml volumes. A2.6.42 Xanthine and Hypoxanthine Agar (Gordon & Mihm, 1957) Prepared as for Tyrosine Agar (Section A2.6.40), but substituting xanthine or hypoxanthine (4 g/1) for tyrosine. A3

MEDIA CONTROL

Quality control in the preparation and evaluation of culture media in the laboratory is essential. This is a continuous process extending from the raw materials, through manufacture to the final product used on the bench. In this Section, some of the basic practicalities and ways of monitoring the suitability of media are considered. The wider aspects of quality control and laboratory proficiency are discussed in Chapter 11. Standardized media are as important as standardized methods and reagents. Commercially prepared media are tested to show that, within certain limits, they conform to stated formulae but, as many of the media contain peptone and other material of biological origin, the analysis may not be particularly helpful and often does not indicate the suitability of the medium for the particular task in hand. In many laboratories, media preparation is still left to junior or unskilled personnel in a 'media kitchen'. However media preparation is far removed from cookery and the work deserves the status of a department. Although media preparation has many features of an art it also has a scientific basis. The aim of quality control is to ensure that media con-

CULTURE MEDIA form to predetermined standards, whereas evaluation implies the determination of their efficacy under the conditions of intended usage. The results of both quality control tests and bacteriological evaluation must be correlated and discrepancies or unexpected results investigated. For example, if after preparation a batch of Glucose-phosphate medium looks yellow (due to overheating) it is not likely to give satisfactory results in the methyl red test and it must be discarded. The growth of Escherichia coli, or other organisms which do not utilize citrate, in Koser's Citrate medium which appears satisfactory on the basis of chemical tests requires further investigation; common causes of such failure include the use of dirty glassware or the presence of organic matter from cotton-wool plugs. And most laboratories have at some time omitted the lactose or bile salts from MacConkey agar, or used a wrong ingredient in media preparation. A3.1

Raw materials

Chemicals, reagents, and carbohydrates should be of Analytical Reagent (AR) quality or conform to Pharmacopoeial standards. The bacteriological laboratory is generally not equipped to carry out the necessary examination of these materials (which may involve spectrophotometry, flame-photometry, ion-exchange, chromatography, titration in non-aqueous media, or potentiometric titration, besides the more conventional assay methods) and it must rely upon the integrity of the supplier. Materials of biological origin are generally more difficult to standardize and may still vary considerably in composition (Report, 1956a). Details of chemical and microbiological assays are outside the scope of this Manual and reference must be made to appropriate monographs and textbooks. A3.1.1 Agar. The concentration necessary to produce a suitable gel varies with the geographical source of the seaweed from which the agar was made (Table A5). A method for estimating the gel strength of agar was described by Jones (1956). A good indication of the source of a sample of agar may be obtained by observation of the diatoms present. These are obtained either by centrifugation of a dilute agar sol or by ashing and extracting the residue with dilute hydrochloric acid; silica skeletons of diatoms, sand particles, and sponge spicules are present in the acid-insoluble ash. Examination of granular or powdered agar is less rewarding as undamaged diatoms are seldom found. Forsdike (1950) examined agars from various sources and his paper illustrates many of the different diatoms. The presence of nitrogenous material may be detected by heating with soda lime, when ammonia is evolved; adulteration with gelatin will be detectable in this way or, alternatively,

by the formation of a turbidity or precipitate when a 1% agar sol is mixed with an equal volume of 1% aqueous picric acid. A3.1.2 Peptone. Although some studies on the constituents of different peptones have been published (Report, 1956a; Habeeb, 1960a, b), much work still remains to be done and until more information is available it is not possible to define standards. All peptones should dissolve completely in water to give clear solutions having a pH value between 5 and 7. Indole production requires a peptone with a high tryptophan content; a gelatin hydrolysate is deficient in tryptophan as is an acid hydrolysate of casein, although an enzymic casein hydrolysate is suitable. Rosenheim's test is of value in detecting tryptophan: to 2 ml 5% peptone solution add 0.05% FeCl3 solution. Cautiously pour concentrated H2SO4 down the side of the tube. A purple colour develops at the junction of the liquids when tryptophan is present. Peptones for use in media for carbohydrate studies must be tested for the presence of fermentable carbohydrates. Soya peptone generally contains carbohydrate and should be avoided in 'sugar' media. The influence of peptone on the methyl red test and carbohydrate reactions are discussed under Evaluation in Section A3.5. It is unlikely that modern peptones will be inhibitory because of a high copper content (O'Meara & Macs ween, 1936, 1937) but this possibility should be borne in mind. A3.1.3 Meat (Beef) extract. In the absence of standards for the product the only simple tests which can be performed are the enumeration of viable organisms (which should be few) and the detection of fermentable carbohydrate (which should be absent). A3.1.4 Yeast extract, unlike meat extract, has a high carbohydrate content. The moisture content may also be high (about 30%) and considerable quantities of NaCl may be present; samples containing more than about 15% NaCl should be rejected. A3.1.5 Bile salts. The complex nature of bile salts makes standardization difficult. Of the components present, deoxycholic acid (as its sodium salt) is widely used (Leifson, 1935). In the absence of suitable chemical standardization, biological evaluation is necessary and we recommend the method described by Burman (1955), in which a batch of bile salt is standardized against a batch known to be satisfactory. The inhibitory effect of bile salts can be influenced by other constituents of the medium such as NaCl (Leifson, 1935) and phosphates (Allen, Pasley & Pierce, 1952). The evaluation of sodium deoxycholate for

209

APPENDIX A use in inhibitory selective media is discussed by Taylor et al. (1964). For some purposes, surfactants such as Teepol or Tergitol can be used instead of bile salts though it should be appreciated that these also are to some extent variable. A3.1.6 Gelatin comes from many animal sources and undergoes different processes; consequently it is available in several grades. For bacteriology, edible gelatin is generally used, the technical grades being unsatisfactory as they often contain preservatives and may have a high SO2 and heavy-metal content. Leffmann & La Wall (1911) considered that a standard should be established for SO2 in gelatin intended for bacteriological use. Some tests for the physical properties of and extraneous matter in gelatin are detailed in a British Standard (757:1959) which gives methods for determining the moisture content, gel strength, viscosity, melting point, water absorption, solubility, keeping quality and pH as well as the amounts of grease, ash, sulphur dioxide, chlorides, arsenic, and heavy metals. A3.1.7 Carbohydrates and related products Testing is usually restricted to the solubility, clarity and pH reaction of solutions, and to simple tests such as absence of reducing sugar in non-reducing sugars, absence of aglycones in glycosides, and the absence of monosaccharides in higher saccharides. Some samples of sucrose may contain invert sugar, detectable by measuring the optical rotation of a solution or more simply by testing with Fehling's or Benedict's solution (Section C1.3) for the presence of reducing substances. Soluble starch should be free from reducing sugars and when treated with iodine solution should give a deep blue colour; a reddish or purple colour would indicate dextrin. Some tests for other carbohydrates are mentioned in Section A3.3.3. A3.1.8 Cole and Onslow's or other pancreatic extracts act by their tryptic content which may be estimated as follows: (i) Qualitative. A 1/100 dilution should digest the gelatin from a used photographic plate or film within 30 minat37 °C. (ii) Quantitative (modified from Douglas, 1922). To 50 ml fresh milk, from which the cream has been removed by centrifugation, add 50 ml 0.2 M-CaCl2; mix thoroughly and measure 5 ml volumes of the milk into 150 x 16 mm (6 x 5/s in) tubes. Place the tubes in a water bath at 38-40 °C. Make dilutions of the enzyme from 1/10 to 1/100 and begin testing with the 1/100 dilution. Add 1 ml of the diluted enzyme preparation to a tube of calcified milk, shake thoroughly, return to the water bath and note the time. Examine the tube at intervals and note the time at which a precipitate or clotting occurs. Continue

210

to test dilutions of the enzyme until the clotting time is between 1 and 2 minutes. Calculation. Suppose 1 ml of a 1/60 dilution of the enzyme clots the milk in 80 seconds, then 1 ml of the original solution contains: (60 x 100)-80 = 75 units. A satisfactory batch of Cole and Onslow's extract should have a tryptic content of at least 50 units per ml. A3.1.9 Thioglycollic acid for inclusion in media should be assayed periodically and rejected when its activity falls below 75% (Report, 1953); the acid should be kept in a tightly stoppered bottle at 4 °C. The stability of thioglycollate solutions has been studied by Cook & Steel (1959a, b) and the assay method of Steel (1958) is recommended. A3.2

Digest media: Intermediate control

During the preparation of digest media (Section A2.1.4) it is important to follow the course of digestion. Samples are taken at the start and at 30-45 minute intervals; the disappearance of undigested protein is most easily shown by the biuret test: heat a 5 ml sample to boiling to stop digestion, cool and add 0.5 ml 10 N-NaOH. Filter and add 0.1 ml 1% CuSO4*5H2O solution to the filtrate. Proteins give a violet colour, proteoses a reddish-pink colour, and amino acids produce no colour reaction. A3.3

Digest media: Final control

The final product of digestion should be examined physically and chemically, as well as by bacteriological tests which are discussed under Evaluation (Section A3.5); some of the causes of faulty media are considered later (Section A3.6). Among the physical properties, colour, clarity, pH, viscosity, and gel strength of solid media should be examined. Determination of specific gravity does not appear to be of much value. For the pH determination of solid media a flat electrode is useful and avoids the need to melt the medium and compensate for the increased temperature. Chemical properties tested may include the presence of the correct ingredients (testing for human error), nitrogen estimations, detection of breakdown products of components (especially those of carbohydrates), and freedom from the end-product(s) which will be looked for after the organism has grown. A3.3.1 Total nitrogen is usually estimated by the Kjeldahl method or some modification of it. In this, organic nitrogen is converted to ammonium sulphate by sulphuric

CULTURE MEDIA acid in the presence of sodium sulphate with copper and selenium as catalysts. The digested reaction mixture is steam-distilled with sodium hydroxide to liberate ammonia. The ammonia is absorbed and estimated colorimetrically with Nessler's reagent or by titration. In all cases a blank determination on the reagents must be made. For details of the technique see an appropriate textbook of analytical chemistry or original papers, for example, by Middleton & Stuckey(1951). Proteose nitrogen is estimated similarly; the proteoses are first separated by precipitation with saturated zinc sulphate solution, and their nitrogen content determined. A3.3.2 Amino nitrogen: one of two methods may be used but the results given by the two methods may not be identical as they do not estimate exactly the same compounds; formol titration is the simpler method but in darkcoloured solutions the end-point may be difficult to see. (i) S0renson 's formol titration method: in this method, formaldehyde reacts with the Nt^-groups of amino acids to form acidic complexes which are titratable with alkali. Neutralize 40% formaldehyde solution (formalin) with 0.1 N-NaOH until just pink to phenolphthalein. To a 5 ml sample of the medium, add 0.05 ml of phenolphthalein solution and 0.1 N-NaOH until just pink; then add 5 ml neutralized formalin and titrate with 0.1 N-NaOH until the pink colour reappears. 1 ml 0.1 N-NaOH = 1.4 mg amino nitrogen. (ii) Pope & Stevens' (1939) method: in this method, copper phosphate reacts with amino acids to form soluble copper complexes; the amount of copper in solution is estimated iodometrically. Copper phosphate suspension: 0.16M-CUC1 2

20 ml

0.18M-Na3PO4 40 ml 0.075 M-borate buffer (pH 8.5) 40 ml Mix the three solutions. The suspension should be freshly prepared and discarded after three days. Place a 5 ml sample of medium in a 50 ml volumetric flask and make just alkaline (blue) to thymolphthalein with 1 N-NaOH. Add one drop of w-octanol to prevent foaming and 30 ml copper phosphate suspension. Make up to 50 ml with distilled water, mix well and filter. Acidify 10 ml filtrate with 0.5 ml glacial acetic acid, add 2 ml of 50% KI solution and 10 ml of 10% KCNS solution. Titrate with 0.01 N-Na2S2O3 using starch mucilage as indicator. The Na2S2O3 is standardized by the same method against 0.01 N-CuSO4. 1 ml 0.01 N-Na2S2O3 = 0.28 mg amino nitrogen.

A satisfactory digest medium should contain about a quarter of its total nitrogen in the form of amino nitrogen. Typical results for the amino acid content of Digest Broth (Section A2.1.4) and Infusion Broth (A2.1.6) are in the range 72-91 and 68-84 mg amino nitrogen per 100 ml respectively. To achieve some degree of standardization, it is usual to adjust the amino nitrogen content of both these media to 70 mg per 100 ml by blending weak and strong batches. The amino nitrogen content of Todd-Hewitt broth (Section A2.6.37) is usually about 117-145 mg per 100 ml. A3.3.3 Other tests. The methods adopted to detect the presence or absence of particular substances in culture media are mainly the straightforward techniques of classical chemical analysis, such as chloride estimation by Mohr titration, but the Extra Pharmacopoeia (1955) is useful for particular tests. Some of the test methods are derived from clinical biochemistry, and a brief selection is given below to illustrate their diversity. Detection of bile salts in MacConkey Broth (Hay's test for bile salts in urine): sprinkle powdered sulphur on the surface of the medium in a wide tube or beaker - bile salts lower the surface tension and the particles sink. Presence of nitrate and absence of nitrite in Nitrate Broth: to 5 ml medium add 1 ml nitrite solution A and 1 ml nitrite solution B (Section C1.14): a red colour indicates nitrite and the batch should be discarded. In the absence of a colour change add 20 mg powdered zinc: the development of a red colour indicates the presence of nitrate. Molisch 's test for carbohydrates: to 5 ml medium add 0.1 ml 5% ethanolic a-naphthol, mix and cautiously pour concentrated H2SO4 down the side of the tube. A purple colour at the junction of the liquids indicates the presence of carbohydrate. Individual carbohydrates may be detected by standard biochemical tests; the distinction between galactose, glucose, lactose and maltose ultimately depends upon the formation of crystalline osazones and their identification by microscopical examination. Several multitests have been proposed by which different carbohydrates can be detected by the same reagent (Barakat & Abd El-Wahab, 1951; Love, 1953); the results of such tests should be confirmed by other methods. Detection of starch in Starch Agar: pour an iodine solution (e.g. Lugol's, Section Bl.l 1) over the medium. A blue colour indicates the presence of starch. Pentoses may be detected by the orcinol reaction: mix equal volumes of test solution and reagent (orcinol, 0.2 g; concentrated HC1, 100 ml; 10% FeCl3, 0.2 ml) and gently heat to boiling. A green or blue-green colour or precipitate indicates the presence of pentoses.

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APPENDIX A Detection of citrate in Citrate media: boil a 5 ml sample of medium with 1 ml mercuric sulphate solution (5% HgO in 20% (v/v) H2SO4), filter, boil, and add 5 drops 1% KMnO4 solution. Decolorization of the reagent and formation of a white precipitate indicates the presence of citrate. Detection ofhippurate in Hippurate Broth: add 5% FeCl3 solution. A brownish-pink precipitate soluble in excess reagent indicates the presence of hippurate. Hydrolysis of salicin in 'sugar media' sterilized by heat treatment is shown by the presence of reducing sugar when the medium is boiled with Benedict's reagent, and by a violet colour when FeCl3 solution is added. Hydrolysis of starch in Starch Serum Water is indicated by a coloured precipitate when a sample of the medium is boiled with Benedict's reagent. Such hydrolysis of starch by the action of amylase may occur when the serum has not been inactivated by heat (Goldsworthy, Still & Dumaresq, 1938). Many of the tests used are not specific but give a good indication of whether the medium has been correctly prepared. In contrast to the bacteriological control methods described below, chemical tests have the advantage of speed. Media in tubes plugged with cotton-wool may lose water by evaporation and, by concentration of ingredients, become unsuitable for their intended use; the addition of sterile water to rectify the concentration is not satisfactory (Vera, 1971). A3.4

Control of equipment

Equipment and apparatus that should be checked at regular intervals include air-filters, autoclaves, hot-air ovens, incubators, water baths, pH meters, deep-freezes, and refrigerators (Vera, 1971), as well as thermometers, other temperature recording devices, filters and filtration techniques, cabinets, anaerobic jars and anaerobic techniques. A3.5

Evaluation of media

It is by the behaviour of media in routine use that the reputation of a media department stands or falls. Sterility is obviously of paramount importance and sterility testing is discussed below. Bacteriological control depends on the nature of the individual medium. Its purposes are to test: (i) the ability of nutrient media to support growth from small inocula; (ii) media used in biochemical tests for their ability to show the desired reactions; (iii) differential media to ensure that organisms growing on them are characteristic; (iv) selective and enrichment media for growth of the organisms wanted and inhibition of other unwanted bacteria.

212

A3.5.1 Sterility testing. A sterility test is a limit test and the conventional technique will detect only gross contamination. The sterility of a product cannot be proved unless the whole of the product (or batch) is tested. Bryce (1956) has calculated that there is a 50% chance of failing to detect contamination when 10 samples are tested from a batch of 500 items, 33 (6.7%) of which are contaminated. However, a contaminant is more likely to develop in a nutrient medium than in a simple aqueous solution, and with bacteriological media it is often sufficient to incubate the medium in order to check for sterility. Culture media are not necessarily suitable for the growth of all micro-organisms; the pH value of a medium intended for bacteria may not be suitable for the growth of fungi. The incubation of an inhibitory medium may fail to show contamination; such a contaminated medium could be the start of a mixed culture which would be revealed only on sub-culture to a non-inhibitory medium. Inhibitory media should therefore be well diluted with sterile Nutrient Broth before incubation. Media likely to be incubated for long periods, such as Dorset Egg and Lowenstein-Jensen, must be free from slow-growing contaminants; however, an adequate turnover is essential as they deteriorate on storage. Many contaminants grow at room temperatures, as do psychrophiles in media stored at low temperatures. In medical bacteriology, however, the presence of thermophiles is much less likely to be a serious problem in media. Theoretically, sterility tests should detect the presence of all living micro-organisms though only bacteria and fungi are usually sought. It is recommended that all samples of media sterilized by filtration or by heat treatment other than adequate autoclaving, should be subjected to the following tests: Incubation temperature 55-60 °C 37 °C 37 °C ambient room (15-20 °C) 4°C

Incubation time 1 days in air 7 days in air 7 days anaerobically 14 days in air 14 days in air

Incubation at 20-25 °C is better than 30-35 °C for showing evidence of inefficient sterilization of materials for injection (Vera, 1971). A3.5.2 Growth-supporting ability. The nature of the medium determines the choice of test organisms; for example with 'Chocolate' Agar, strains of Haemophilus and Neisseria species should be used, whereas with Nutrient Agar, less exacting organisms, such as staphylococci and coliform organisms are suitable. The organisms should be cultured in a suitable medium overnight and nine 10-fold serial dilutions prepared in sterile quarter-strength Ringer's

CULTURE MEDIA solution; 0.02 ml of each dilution is placed on the surface of medium contained in a Petri dish (as in the Miles & Misra (1938) surface-viable counting technique) or added to 5 ml volumes of liquid media. After incubation, the greatest dilution giving growth is noted. The ability of media to support the growth of anaerobic bacteria is determined in a similar manner. Clostridium perfringens is not a good test organism for anaerobiosis; more suitable species are C. chauvoei (very exacting), C. novyi, or C. tetani, but the film-like growth of C. tetani may be difficult to see. Some fastidious anaerobes (e.g. Bacteroides; Streptobacillus moniliformis) may be inhibited by oxidized reducing agents or by Liquoid (sodium polyanethol sulphonate) in media (Holdeman & Moore, 1972). Messer (1947) found an inhibitory agent in a batch of Nutrient Agar and traced it to formaldehyde used to increase the wet strength of filter paper used during the preparation of the medium. With MacConkey Agar, suitable test organisms include Escherichia coli, Salmonella typhimurium, Shigella dysenteriae, and Shigella sonnei. In addition to assessing its growth-promoting ability, this test also serves to show whether the medium distinguishes satisfactorily between these enterobacteria. MacConkey Broth should be tested for its ability to show acid and gas formation at 44 °C when inoculated with a strain of E. coli. A3.5.3 Biochemical performance. Samples of media must be tested with organisms known to produce positive and negative reactions. Examples of such control organisms are given under the individual test methods in Appendix C. Because of strain variation within species, it is essential that the particular strain used for test purposes possesses the desired characters and in Table Dl we list suitable strains, maintained in the National Collection of Type Cultures, of the organisms required. The need for biological control was shown by Orr Ewing & Taylor (1945) who found that certain batches of peptone were unsuitable for making carbohydrate media as only in some of them did the Newcastle and Manchester biotypes of Shigella flexneri 6 produce both acid and gas. Peptone may also affect the results of a finely balanced reaction such as that of the MR (methyl red) test; each batch of peptone should therefore be tried out in tests with known MRpositive and -negative strains (Jennens, 1954). A3.5.4 Differentiation. Differential media must be tested to make sure that organisms growing on them exhibit the

typical characters by which they are differentiated from other organisms. For example, the testing of MacConkey Agar for its ability to distinguish lactose-fermenting and lactose-non-fermenting bacteria has been noted above. Tellurite media should be inoculated with each of the three varieties of Corynebacterium diphtheriae and after incubation these should be examined for their characteristic colonial appearances. A3.5.5 Selectivity. A selective or an enrichment medium should be able to support the growth of a particular organism or group of organisms, and at the same time inhibit others. A convenient method of testing this is to mix broth cultures of the organism to be selected with another organism to be inhibited, in varying proportions (e.g. 1:2, 1:4 and so on). A loopful of each mixture is plated on solid media or inoculated into liquid media. After incubation an estimate of the selectivity of a solid medium may be made by inspection of the growth. The efficiency of a liquid enrichment medium cannot be judged until a subculture to a noninhibitory solid medium has been made. A3.6

Causes of faulty media

Major sources of trouble include errors in weighing and measuring, inadvertent use of the wrong ingredients (e.g. hydrated instead of anhydrous salts), and incorrect pH adjustment. Some common faults and their possible causes are listed below: Loss of growth-promoting capacity Over-sterilization; repeated remelting of solid media; burning or charring; contamination with metallic salts; incorrect molarity due to careless pH adjustment. Decreased gel strength Over-sterilization; hydrolysis of agar media of low pH; incomplete solution of agar; repeated remelting. Darkening Over-sterilization; caramelization of sugars; local superheating due to inadequate mixing. pH change Over-sterilization; incomplete mixing; use of alkaline containers; hydrolysis of ingredients; repeated remelting. Precipitation Chemical incompatibility; failure to remove phosphates; over-sterilization; prolonged holding of melted agar media at high temperature.

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APPENDIX B Staining: reagents and methods

REAGENTS

Bl

All staining reagents should be kept in well-closed glassstoppered bottles (except Loeffler's methylene blue) and protected from direct sunlight. For frequent use, flexible plastic bottles with tube outlets may be used but they must be washed out thoroughly before refilling. They should not be stored in close proximity to concentrated acids or ammonia. Distilled water for reagents should be freshly prepared and neutral in reaction. Formulae of staining reagents are listed in alphabetical order. Bl.l

Acetone - iodine solution for decolorization

Strong iodine solution Iodine 10 g Potassium iodide 6g Distilled water 10 ml to 100 ml Ethanol (90%) Dissolve the iodine and potassium iodide in the water and adjust to volume with the ethanol. Acetone-iodine mixture Strong iodine soln Acetone Mix well before use

3.5 ml 96.5 ml

Dissolve the dyes in the ethanol. Mix the acid with the water and add to the dye solution. Allow to stand for 24 h and then filter. B1.4

AgNO3 5g Distilled water 100 ml Dissolve the nitrate in the water; to 90 ml of this solution add strong ammonia solution (sp. gr. 0.880) drop by drop until the precipitate which forms just dissolves; add sufficient of the remaining AgNO3 solution drop by drop until the reagent remains faintly turbid even after shaking. When protected from light, this reagent is stable for several weeks. B1.5

Acid-alcohol

Cone. HC1 Ethanol (95%) Mix well before use. B1.3

3 ml 97 ml

Albert's stain

Malachite green Toluidine blue Ethanol (95%) Glacial acetic acid Distilled water

214

0.2 g 0.15 g 2 ml 1 ml 100 ml

Ammonium oxalate - crystal violet stain

Solution A Crystal violet 10 g Ethanol (95%) 100 ml Mix and dissolve. Solution B 1 % aq. soln Ammonium oxalate For use, mix 20 ml of solution A and 80 ml of solution B. B1.6

B1.2

Ammoniacal silver nitrate solution

Aqueous solutions

Simple aqueous solutions of each of the following are used in staining. Bismarck brown 0.2 % Chrysoidin 0.4 % Malachite green 0.5 % Malachite green 5% Safranin 0.5 % B1.7

Carbol fuchsin

Carbol fuchsin, strong Phenol

85 g

STAINING Basic fuchsin 15 g Ethanol 250 ml Distilled water 1250 ml Mix the phenol and fuchsin and if necessary heat gently to dissolve the phenol. Add the ethanol and distilled water and filter into a stoppered bottle. Propanol may be substituted for ethanol. Carbol fuchsin, weak Dilute one volume of strong carbol fuchsin with 10-20 volumes of distilled water. B1.8

Giemsa stain: stock solution

Giemsa stain (powder) 1.0 g Glycerol 60 ml Absolute methanol 60 ml Heat the glycerol to 55-60 °C in a water bath. Add the stain and mix thoroughly. Incubate the mixture at the same temperature for 2 hours. Allow to cool and add the methanol. This stain is best used after 'maturation' on the shelf for about 2 weeks. For use, add 1 part of stock solution to 10 parts of 0.01 M-phosphate buffer (pH 7.0) and allow to stand for 30 minutes. B1.9

India ink

A dense, homogeneous India ink free from large particles or clumps of particles is necessary. Duguid's (1951) practice is to mix the ink with a quarter of its volume of grade12 Ballotini glass beads (0.22 mm) and shake for 1 h in a Mickle tissue disintegrator. Thin ink may be improved by evaporation to concentrate it. B1.10

Kirkpatrick's fixative

Absolute ethanol Chloroform Formaldehyde (40%) soln Mix well before use. B1.11

60 ml 30 ml 10 ml

Loeffler' s methy lene blue

(Loeffler, 1884) Saturated ethanolic solution Methylene blue Ig 100 ml Ethanol (95%) Staining solution KOH l%aq. soln 1 ml 99 ml Distilled water 30 ml Ethanolic methylene blue ition, a Mix in order; this reagent must be ripened by oxidation, ing can process taking several months to complete, but ripening

be hastened by aeration. Bottles should not be more than half-full, the stopper replaced by a light cotton-wool plug and the bottle shaken frequently. The stain improves with keeping and batches sufficiently large to last for 5-10 years may be prepared Note. The ripened stain is sometimes called polychrome methylene blue. B1.12

Lugol's iodine

Iodine 5g Potassium iodide 10 g Distilled water 100 ml Dissolve the iodide and iodine in some of the water, and adjust to 100 ml with distilled water. Note. This reagent is Aqueous Iodine Solution of the British Pharmacopoeia (1963). For use dilute 1/5 with distilled water. B1.13

Muir's mordant

HgCl2, saturated aq. soln (about 7%) Potash alum, saturated aq. soln (about 12%) Tannic acid, 20% aq. soln Mix well before use. B1.14

20 ml 50 ml 20 ml

Neisser's stain

Solution A Methylene blue 0.1 g Ethanol (95%) 5 ml Glacial acetic acid 5 ml Distilled water 100 ml Dissolve the dye in the water and add the acid and ethanol. Solution B Crystal violet 0.33 g Ethanol (95%) 3.3 ml Distilled water 100 ml Dissolve the dye in the ethanol-water mixture. For use, mix 20 ml of solution A and 10 ml of solution B. B1.15

Ryu's flagella stain

(Kodaka, Armfield & Lombard, 1982) Solution A (mordant) Tannic acid (powdered) Phenol 5% aq. soln Aluminium potassium sulphate (12-hydrate) saturated soln Solution B (stain) Crystal violet

215

10 g 50 ml 50 ml 12 g

APPENDIX B Ethanol 100 g (a saturated solution) Mix 10 parts of solution A with 1 part solution B and store at ambient temperature. The mixture is stable indefinitely and does not require filtration before use. Allow to stabilize by standing for 2-3 days as the freshly prepared stain is often too potent. As a rule of thumb, apply a fairly fresh stain for 5 min, or a stabilized stain for 10 min. B1.16

Plimmer's mordant

Tannic acid 20 g A1C13-6H2O 36 g ZnCl2 20 g Basic fuchsin 3g Ethanol (60%) 80 ml Grind the solids together in a mortar and add the ethanol; triturate until dissolved. Before use, add 1 volume of mordant to 3 volumes of distilled water and mix well. B1.17

Rhodes' mordant

Tannic acid, 10% aq. soln 60 ml Potash alum, saturated aq. soln (about 12%) 30 ml Aniline, saturated aq. soln (about 3.5%) 6 ml FeCl3, 5% aq. soln 6 ml Add the alum solution to the tannic acid solution, followed by the aniline solution. Redissolve the curd which forms by shaking. Add the FeCl3 solution and allow the resulting black solution to stand for 10 min before use. B1.18

Sudan Black

Sudan black B powder 0.3 g 70% ethyl alcohol 100 ml Shake well and stand overnight before use. Keep in a wellstoppered bottle. B2

METHODS

In this section, we describe a small selection of staining methods which are usually satisfactory; for a more complete coverage, reference must be made to specialist monographs such as those by Conn, Darrow & Emmel (1960), E. Gurr (1956) and G.T. Gurr (1963). After microscopical examination, used slides should be sterilized by autoclaving. It must never be assumed that bacteria will have been killed by the application of the staining reagents. Anthrax spores, for example, have germinated in the immersion oil left on a slide, and Soltys (1948) reported cutaneous anthrax in a veterinary student infected from a stained film.

216

B2.1

Simple stains

Loeffler's methylene blue is perhaps the most valuable reagent available for staining bacteria. It is excellent for bacteria of the genus Corynebacteriwn where beading, barring, and granules may be demonstrated, especially when the organism has been grown on Loeffler serum slopes. With sporing organisms stained with this reagent, the spores appear as unstained bodies within blue cells. 1. Stain for 1 minute. 2. Rinse with water. 3. Drain or blot to dry. B2.2

Differential stains

B2.2.1 Gram's method. In the USA the method given here is often referred to as Hucker's modification (Hucker & Conn, 1923), and in this country as Lillie's (1928) modification. 1. Apply ammonium oxalate-crystal violet stain for xh min. 2. Wash in water. 3. Apply Lugol's iodine solution for xh min. 4. Drain off the iodine solution but do not wash. 5. Decolorize with a few drops of acetone. (Note: acetone decolorizes very quickly and should not be left on the film for more than 2-3 seconds). 6. Wash thoroughly in water. 7. Counterstain with 0.5% safranin for xli min. (A few Gram-negative organisms such as H. influenzae and Y. pestis do not stain well with safranin. Films for such organisms should be counterstained with weak carbol fuchsin for lh min.) 8. Wash and stand slide on end to drain, or blot dry. Preston & Morrell (1962) modified Lillie's method by retarding decolorization with an acetone-iodine mixture. 1. Apply ammonium oxalate-crystal violet stain for 72 min. 2. Wash off thoroughly with Lugol's iodine soution. 3. Apply Lugol's iodine for 72 min. 4. Wash off thoroughly with acetone-iodine mixture. 5. Apply acetone-iodine solution for ]/i min. 6. Wash thoroughly with water. 7. Counterstain with weak carbol fuchsin for 72 min. 8. Wash and drain or blot to dry. The whole slide must be flooded with each reagent and the previous reagent must be completely removed at each stage. Insufficient reagent may result in uneven staining or decolorization. Gram-positive organisms are blue or purple; Gram-negative organisms are red. Some workers prefer to counterstain with Bismarck brown, which they claim gives a better contrast.

STAINING B2.2.2 Ziehl-Neelsen's method (Acid-fast stain) (Ziehl, 1882; Neelsen, 1883) 1. Flood the slide with strong carbol fuchsin and heat until steam rises (but do not boil). 2. After 3 ^ min apply more heat until steam rises again; do not let the stain dry on the slide. 3. About 5-7 min after the first application of heat wash the slide thoroughly under running water. 4. Decolorize in acid-alcohol until all traces of red have disappeared from the film. Decolorization should not be attempted in one stage; there should be intermittent washings in water and re-application of acid-alcohol. 5. Wash well in water when decolorization is complete. 6. Counterstain with Loeffler's methylene blue or 0.5% malachite green for 1 min. 7. Wash and stand on end to drain; DO NOT BLOT. Acid-fast organisms are red; other organisms are blue or green. Numerous variations have been advocated such as cold staining or the addition of detergents to the fuchsin but they have little if any advantage over the conventional ZiehlNeelsen method. B2.3

Special stains

B2.3.1 Spore stains Method 1 (modified from Moeller 1891) This is similar to Ziehl-Neelsen's method but ethanol is used for decolorization. 1. Flood the slide with strong carbol fuchsin and steam. 2. After 5 min wash well in water. 3. Decolorize with ethanol until all traces of red have been removed. Decolorization of the vegetative bacilli can best be controlled by examining the wet film under low power microscopy (after the bottom of the slide has been wiped dry). 4. Wash thoroughly in water. 5. Counterstain with Loeffler's methylene blue for 1-2 min. 6. Wash and drain or blot to dry. Bacterial bodies stain blue, spores red. Method 2 (Schaeffer & Fulton's 1933) 1. Flood the slide with 5% aqueous malachite green and steam for 1 min. 2. Wash under running water. 3. Counterstain with 0.5% aqueous safranin for 15 seconds. 4. Rinse with water and drain or blot to dry. Bacterial bodies stain red, spores green. This method can be used as a cold stain by allowing the malachite green to act for 10 min.

B2.3.2 Capsule stains Muir's method 1. Flood with strong carbol fuchsin and heat gently for 1 min. 2. Rinse rapidly with ethanol, then wash well in water. 3. Flood with Muir's mordant (B1.13) for 30 seconds. 4. Wash off with water, and then with ethanol for 30 seconds or until the film is pale red. 5. Wash well with water. 6. Counterstain with Loeffler's methylene blue for 30 seconds. 7. Wash and drain or blot to dry. Bacteria stain red, capsules blue. Giemsa method To fix, immerse air-dried films for 3 minutes in absolute methanol or 1:1000 mercuric chloride. Immerse slide in Giemsa stain (B1.8). Wash with 0.01 M-phosphate buffer, pH 7.0 for 30 seconds, blot and allow to dry. Examine under oil immersion lens. Bacterial cells appear as bluepurple rods surrounded, if present, by pink-red capsules. India ink wet-film method (Duguid, 1951) This is a 'negative' stain, which is not a stain in the true sense of the word but a means of colouring the background so that the cells are shown in relief as clear objects. 1. Place a large loopful of undiluted India ink on a slide. 2. Mix this with a small portion of the bacterial colony or a small loopful of the deposit from a centrifuged liquid culture. 3. Place a coverslip on top and press down under a pad of blotting-paper. Ideally, the film should be of the same thickness as the capsulate organisms. If the coverslip is not pressed down sufficiently, the organisms will tend to drift in the ink and may be obscured by overlying ink; if pressed down too much, the capsules may be distorted. The capsule appears as a clear light zone between the refractile cell outline and the dark background. For demonstrating slime production by enteric organisms, Duguid cultured them on excess Sugar Agar, a medium containing maltose, phosphate, mineral salts, and a low concentration of peptone. B2.3.3 Stains for metachromatic (volutin) granules For metachromatic granules make a smear from a culture on Loeffler Serum medium. Albert's stain, modified (Albert, 1921; Laybourn, 1924) 1. Stain with Albert's stain (B1.3) for 3-5 min. 2. Wash with water and blot to dry. 3. Stain with Lugol's iodine solution for 1 min. 4. Wash with water and drain or blot to dry.

217

APPENDIX B Cytoplasm appears light green, granules blue-black. Neisser's (1903) stain 1. Stain with Neisser's stain (B1.14) for 10 seconds. 2 Rinse rapidly with water. 3. Stain with 0.2% Bismarck brown or 0.4% chrysoidin for 30 seconds. 4. Wash rapidly in water, drain or blot dry. Cytoplasm appears light brown, granules blue-black. B2.3.4 Flagella stains. The arrangement of flagella on the bacterial cell has long been used as a taxonomic criterion. Unfortunately it is not one which is easily or unequivocally determined. The most satisfactory method of determining the mode of bacterial flagellation is by electron microscopy but, since a two-dimensional picture of a threedimensional object is obtained, the electron micrographs must be interpreted with caution. Flagella staining should not be undertaken lightly, though reasonably satisfactory results can be obtained when care is taken in the preparation of the culture, the film, and the reagents. Known peritrichous and polar flagellate organisms should be used as controls. The interpretation of the results of flagella staining has been discussed briefly by Hodgkiss (1960). For staining flagellae, Ryu's method modified by Kodaka, Armfield & Lombard, (1982) is recommended. We also repeat the flagella stains given in the previous edition of this Manual for those experienced in their use. Use only young cultures grown under optimum conditions for the organism under study on agar media that do not contain fermentable carbohydrates. Touch a colony with the tip of a straight inoculation wire, taking care not to pick up any agar, then dip the wire in the centre of each of 2 drops of distilled water on a microscope slide. The slide should have been previously cleaned by heating in a (blue) Bunsen flame so that the drops of water wet the surface easily and

218

spread out spontaneously. There should be no turbidity the aim is to transfer only a few bacteria. Leave the preparation to dry without disturbance. After staining, examine microscopically from the periphery of each spot inwards in order to find the areas of optimum staining. The deposition of coarse blobs of stain all over the slide indicates overstaining. Ryu's method, modified (Kodaka, Armfield & Lombard, 1982) 1. Apply Ryu's stain (B1.15) to the entire slide for 5 min (fresh stain) or 15 min (matured stain). 2. Wash in running water and allow to dry. Cesares-Gill's method, modified (Plimmer & Paine, 1921) 1. Treat with Kirkpatrick's fixative (B1.10) for 5 min. 2. Wash off the fixative thoroughly with water. 3. Filter the diluted Plimmer's mordant (B1.16) onto the slide and allow to act for 5 min. 4. Wash off with water. 5. Stain for 2 min with weak carbol fuchsin. 6. Wash and allow to dry in air. Rhodes' (1958) method This is a modification of Fontana's method of staining spirochaetes. 1. Apply Rhodes' iron tannate mordant (B1.17) for 3-5 min. 2. Thoroughly wash with water. 3. Heat the ammoniacal silver nitrate solution nearly to boiling and apply to the film. 4. Leave to act for 3-5 min. 5. Wash well with water. 6. Drain or blot to dry. Unless a permanent preparation is made, the stained film disintegrates after about a week on exposure to air.

APPENDIX C Characterization tests Cl Reagents C2 Buffer Solutions C3 Test Methods

Cl

REAGENTS

Formulae of and notes on the reagents are shown below. Standard acids and alkalis, and simple aqueous solutions are not listed. Cl.l

Acid ferric chloride

12 g FeCl3-6H2O Cone. HC1 2.5 ml Distilled water to 100 ml Dilute the acid with 75 ml of the water, dissolve the ferric chloride by warming gently, and adjust to volume with water. C1.2

Acid mercuric chloride (Frazier, 1926)

12 g HgCl2 Distilled water 80 ml Cone. HC1 16 ml Mix the mercuric chloride with the water, add the acid, and shake well until solution is complete. Note. For reasons of safety, 30% trichloracetic acid may be used in tests for gelatinase and caseinase activity instead of the acid mercuric chloride solution. C1.3

C1.4

Creatine solution

1% creatine in 0.1 N-HC1 This solution avoids the rather vague 'knife-point of creatine' quoted in many textbooks and is relatively stable; a solution of creatine in alkali is often recommended, but is unstable (Levine et al., 1934). Ethylhydrocuprein (EHC) discs (synonym: Optochin discs), see Section Cl.l8. C1.5

Ehrlich's reagent

p-dimethylaminobenzaldehyde 1 g Ethanol, absolute 95 ml Cone. HC1 20 ml Dissolve the aldehyde in the ethanol and add the acid. Protect from light. C1.6

Hydrogen peroxide

H2O2, 3% aq. soln ('10% volume') Protect from light and store in a cool place. Keep in a brown bottle closed with a glass stopper, paraffined cork or plastic screw-cap. Peroxide paper test strips are also available commercially.

Benedict's qualitative solution

Sodium citrate 17.3 g Na2CO3, anhydrous 10 g CuSO4-5H2O 1.73 g Distilled water to 100 ml Dissolve the sodium citrate and carbonate in 60 ml of the water. Dissolve the copper sulphate in 20 ml of the water and add it, with constant stirring, to the first solution. Adjust to volume with water. Note. The solution should be stored in a warm place as it is liable to crystallize if cold.

C1.7

Kovacs' reagent for indole (Kovacs, 1928, 1956)

/7-dimethylaminobenzaldehyde 5g Amyl alcohol 75 ml Cone. HC1 25 ml Dissolve the aldehyde in the alcohol by gently warming in a water bath (about 50-55 °C). Cool and add the acid with care. Protect from light and store at 4 °C. Note. The reagent should be light yellow to light brown in colour; some samples of amyl alcohol are unsatisfactory,

219

APPENDIX C and give a dark colour with the aldehyde. A few organisms produce indole weakly and the use of Ehrlich's instead of Kovacs' reagent increases the sensitivity of the test. For micromethods this reagent is prepared with iso-amyl alcohol. Lugol's iodine solution: see B 1.12. C1.8

Lytic enzyme for streptococcal grouping (Maxted, 1948).

Streptomyces griseus NCTC 7807, originally stated by Maxted to be Streptomyces albus, is grown on a suitable medium (Section A2.6.36) in Roux bottles for 4 to 5 days at 37 °C. After growth, the agar gel is destroyed by freezing in solid CO2. The fluid which exudes on thawing contains the lytic principle and can be sterilized by filtration but this is not essential; the pH is not adjusted, nor is a preservative added. Store in a refrigerator. C1.9

Methyl red solution

Methyl red 0.04 g Ethanol 40 ml Distilled water to 100 ml Dissolve the methyl red in the ethanol and dilute to volume with the water. C1.10

oc-naphthol solution

5% a-naphthol in ethanol (not 95% ethanol) The solution should not be darker than straw colour; if necessary the a-naphthol should be redistilled (Fulton, Halkias & Yarashus, 1960). Cl.ll

Nessler's reagent

Dissolve 5 g potassium iodide in 5 ml freshly distilled water. Add cold saturated mercuric chloride solution until a slight precipitate remains permanently after thorough shaking. Add 40 ml 9 N-NaOH. Dilute to 100 ml with distilled water. Allow to stand for 24 h. Alternative formula: dissolve 8 g potassium iodide and 11.5 g mercuric iodide in 20 ml water and adjust to 50 ml. Add 50 ml 6 N-NaOH. Mix and allow to stand for 24 h. Notes. The water used in its preparation must be ammonia-free. Allow the reagent to settle before use. Protect from light.

220

C1.12

Niacin test reagents

(i)

4% aniline in 95% ethanol OR 1.5% o-tolidine in 95% ethanol (ii) 10% aq. cyanogen bromide Store at 4 °C for up to 2 weeks. Cyanogen bromide solution is toxic and must be treated with an equal volume of ammonia solution (sp. gr. 0.880) or 10 N-NaOH before disposal. C1.13

Ninhydrin reagent (for hippurate hydrolysis Method 3).

Dissolve 0.35 g ninhydrin in 10 ml of a 1:1 mixture of acetone and butanol. Prepare freshly for each batch of tests. Alternatively, dispense in bijoux bottles and store in the dark (C3.27, Method 4). C1.14

Nitrite test reagents

Solution A 0.33% sulphanilic acid in 5 N-acetic acid Dissolve by gentle heating. Solution B 0.6% dimethyl-a-naphthylamine in 5 N-acetic acid OR 0.5% a-naphthylamine in 5 N-acetic acid OR 0.13% 1-naphthylamine-7-sulphonic acid (Cleve's acid) in 5 N-acetic acid Dissolve by gentle heating. Zinc dust or 10% zinc dust suspended in 1% methylcellulose solution (Steel & Fisher, 1961) Note. Cleve's acid is preferred to a-naphthylamine, which is regarded as carcinogenic. C1.15

Oxidase test reagents

Solution 1 Prepare a fresh solution of tetramethyl-/?-phenylenediamine dihydrochloride each time of use by adding a loopful of it to about 3 ml of sterile distilled water or saline. Do not use if it becomes blue. Autoxidation of the reagent occurs rapidly and although this can be retarded by the addition of 1% ascorbic acid, it is not sufficiently stable in aqueous solution for storage. Solution 2 1% a-naphthol in 95% ethanol

CHARACTERIZATION Solution 3 1 % /7-aminodimethylaniline oxalate aq. soln (also known as dimethyl-p-phenylenediamine oxalate) As with solution 1, autoxidation may occur; if ascorbic acid is not used to delay the process the solution should be freshly prepared each week and kept at 4 °C. C1.16

Sodium hippurate solution (1 %)

Sodium hippurate 0.1 g Sterile distilled water 10 ml Mix in sterile tubes and place 0.4 ml aliquots in sterile bijoux bottles. Freeze at -20 °C until required. C1.17

Test papers with reagents

Cut filter paper strips 5-10 mm wide and 50-60 mm long, and impregnate with the appropriate solution; dry at 50 to 60 °C. Store in screw-capped containers. For the detection ofH2S (Clarke, 1953a) Lead acetate hot saturated aq. soln (10 g lead acetate in 100 ml hot water) Note: for the micromethod the papers should be 4 to 5 mm wide. For the detection ofindole (Holman & Gonzales, 1923) Oxalic acid, hot saturated aq. soln For the detection ofindole (Gillies' 1956) p-dimethylaminobenzaldehyde 10 g
2,4-diamino-6,7-di-/s<9propyl pteridine phosphate* 0.1 g acetone 100 ml Note. 0/129 discs containing 10 jug and 150 \xg of pteridine phosphate are available commercially. For sensitivity to optochin 0.05% ethylhydrocuprein hydrochloride aqueous solution. For sensitivity to bacitracin Discs 6 mm in diameter containing 0.1 unit of bacitracin should produce zones of growth inhibition of at least 12 mm with Streptococcus pyogenes and Streptococcus pneumoniae. For sensitivity to Liquoid Discs containing 1 mg of the anticoagulant sodium polyanethol sulphonate (Liquoid) give wide zones of inhibition with Peptostreptococcus anaerobius and Streptobacillus moniliformis. For sensitivity to metronidazole Discs containing 5 jig metronizadole give wide zones of inhibition with many anaerobic organisms. Pyridoxal requirement The requirement for this growth factor can be demonstrated with discs containing 20 JLLI of a solution (1 mg/ml) on a medium that does not contain yeast extract: for example Blood Agar prepared with a Columbia base. Cysteine requirement The requirement of some streptococci and other organisms for cysteine over and above that present in ordinary culture media can be tested with discs containing 10 JLXI of a 40% (w/v) solution of cysteine hydrochloride. For X-, V-, and X+ Vfactor requirement Discs soaked with solutions of X-, V-, or a mixture of X- and V-factors are used for confirmation and identification of haemophili. C1.19

Tween 80-peroxide mixture (Kubica, 1973)

10% Tween 80 (10%) made by adding 10 ml Tween to 90 ml distilled water. Sterilize at 121 °C for 10 min. If the Tween separates, rotate the flask to ensure mixing. Before use mix equal volumes of 10% Tween 80 and 30% H7O,.

Sensitivity discs and test papers

Sterile filter paper discs 6 to 8 mm in diameter (e.g. Whatman No. 3) are soaked in the appropriate solution, drained, and dried at 37 °C. Dried discs can be stored in small screw-capped bottles and should be kept at 4 °C. Several of them are available commercially. For sensitivity to O/129 (Bain & Shewan, 1968)

* Obtainable from BDH Chemicals Ltd., Broom Road, Poole, Dorset BH12 4NN, UK. Pteridine phosphate (Cat. No. 44169); Peroxide test (Cat. No. 31520 2F).

221

APPENDIX C C1.20

Preparation of X- and V-factors (Marshall & Kelsey, 1960)

X-factor. Centrifuge the red cells from 40 ml blood and add to them with shaking 100 ml acetone containing 1.2 ml cone. HC1; filter and add 100 to 120 ml water to the filtrate to precipitate the haemin. Collect by filtration and wash with water. Dissolve the crude haemin in 25 ml 0.1 MNa2HPO4 and sterilize at 115 °C for 10 min. V-factor. Suspend 50 g yeast in 100 ml 0.2 M-KH2PO4 and heat at 80 °C for 20 min. Clarify by centrifugation and sterilize the supernatant by filtration. Store in a refrigerator or deep-freeze. C2

BUFFER SOLUTIONS

All traditional and biological buffer solutions should be stored in well-stoppered plastic or alkali-free glass bottles. The solutions should not be used for more than 2-3 months after preparation; chemically clean bottles should be used for fresh solutions. C2.1

Phthalate buffer, 0.0125 M, pH 5.0

Solution A 0.05 M-potassium hydrogen phthalate (C6H4(COOH) COOK, mol. wt = 204.23) Solution B 0.05 N-NaOH (carbonate-free) To prepare, mix as follows: soln A soln B pH5.0 50 ml + 23.85 ml Dilute to 200 ml with distilled water. In the micromethod for decarboxylases, 5 ml 1 % ethanolic bromcresol purple is added to 200 ml buffer. C2.3

C2.4

S0rensen's citrate buffer, 0.01 M, pH 5.6

Solution A 0.1 M-citric acid (C6H8O7H2O, mol. wt = 210.15) Solution B 0.1 M-fr/-sodium citrate (Na3C6H5O7-2H2O, mol. wt = 294.11) To prepare the buffer, mix as follows: solnB soln A pH5.6 36.3 ml 13.7 ml dilute to 500 ml with distilled water.

Mcllvaine's buffer

Solution A 0.1 M-citric acid (C6H8O7H2O, mol. wt = 210.15) Solution B 0.2 M-disodium phosphate (Na2HPO4, mol. wt= 141.97) To prepare the buffer, mix as follows: soln A solnB pH4.0 61.45 ml + 38.55 ml pH 6.0 36.85 ml + 63.15 ml C2.2

(Na2HPO412H2O, mol. wt = 358.16) Solution B 0.025 M-KH2PO4 (mol wt = 136.09) To prepare the buffer, mix as follows: soln A soln B pH6.0 12 ml + 88 ml pH6.8 50 ml + 50 ml For the malonate and urease micromethods, 5 ml 0.1% ethanolic phenol red is added to 100 ml buffer.

Phosphate buffer, 0.025 M, pH 6.0

Solution A 0.025 M-Na2HPO4

222

C3

CHARACTERIZATION TEST METHODS

Unless otherwise indicated, cultures are incubated at 37 °C or at their optimum temperature for growth. Recommended organisms for use as controls are given in Appendix D. C3.1

Acetylmethylcarbinol production; the Voges-Proskaiier reaction.

Method 1 (Barritt, 1936). After completion of the methyl red test (C3.35) add 0.6 ml 5% a-naphthol solution (C1.10) and 0.2 ml 40% KOH aqueous solution; shake well, slope the tube (to increase the area of the air-liquid interface), and examine after 15 min and 1 h. A positive reaction is indicated by a strong red colour. Method 2 (O'Meara, 1931). After completion of the methyl red test, add 2 drops (about 0.05 ml) creatine solution (C1.4) and 1 ml 40% KOH aqueous solution); shake, slope, and examine after 1 and 4 h. A positive reaction is indicated by an eosin-pink colour. Method 3 (Furniss, Lee & Donovan, 1978). Stab-inoculate semi-solid VP medium (A2.5.11); incubate for 1-3 days. Place on the surface 1 drop creatine solution (C1.4) and about 0.5 ml freshly prepared mixture of 3 parts 5% anaphthol (C1.10) and 1 part 40% KOH; shake gently to aerate, and read after 1 h. A positive reaction gives a red colour. Note. Motility can also be observed with this medium

CHARACTERIZATION after incubation; motile strains show diffuse growth whereas with non-motile organisms, growth is limited to the line of the inoculum. Method 4 (NCTC micromethod; Clarke & Cowan, 1952). Incubate the culture overnight on 1% Glucose Agar; wash off with water or saline; centrifuge and resuspend the deposit in water or saline so that the density corresponds approximately to 109 Escherichia coli per ml. Test: using capillary pipettes that deliver 50 drops/ml, pipette into small tubes (e.g. 65 x 10mm) as follows: 10% glucose 1 drop (0.02 ml) 1 drop (0.02 ml) 0.2% creatine 0.025 M-phosphate buffer, pH 6.8 2 drops (0.04 ml) Suspension from Glucose Agar 2 drops (0.04 ml) Incubate in water bath at 37 °C. Test after 2 h. Add 3 drops (0.06 ml) 5% cc-naphthol in ethanol (CLIO); shake well to mix; then add 2 drops (0.04 ml) 40% KOH, shake, leave on bench and after 10 min read the result. A positive reaction is indicated by a red or pink colour. Controls for all methods: positive - Enterobacter cloacae; Streptococcus anginosus ('milleri'); Vibrio alginolyticus negative- Escherichia coli; Streptococcus pyogenes; Vibrio parahaemolyticus Acid production from sugars: see Carbohydrate breakdown (C3.8.1). C3.2

Aesculin hydrolysis

Inoculate Aesculin Broth and examine daily up to 5 days for blackening; this indicates hydrolysis of the aesculin. Alternatively, inoculate Aesculin Agar and look for blackening in and around the bacterial growth. Controls: positive - Enterococcus faecalis negative - Streptococcus agalactiae C3.3

C3.5

Bile solubility

Method 7. To 5 ml of an 18 hour culture of the test organism in Serum, Digest, or Infusion Broth add 0.5 ml 10% sodium deoxycholate solution and incubate at 37 °C. Pneumococci are lysed within 15 min of adding the bile salt. Note. A glucose-containing medium is unsatisfactory for this test as the reaction should not be more acid than pH 6.8. Method 2. Grow the organism in Serum Broth or ToddHewitt broth for 24 h. Centrifuge and discard the supernatant. Resuspend the organisms in phosphate buffered saline (pH 7.3). Add 0.5 ml 10% sodium deoxycholate solution and incubate at 37 °C for 15-30 min. Under these conditions, rapid clearing of the suspension occurs with pneumococci and an occasional strain of E. faecalis; if clearing has not taken place in 30 min the organisms are not pneumococci. Method 3 (Hawn & Beebe, 1965). For a rapid presumptive test on the primary plate touch a suspected colony with a loop charged with 2% sodium deoxycholate (pH 7.0) solution. Incubate the plate for 30 min at 37 °C. Colonies of pneumococcus disappear and, on Blood Agar, leave an area of a-haemolysis. Controls: positive - Streptococcus pneumoniae negative - Streptococcus agalactiae

Aesculin-bile test for streptococci

Streak- or spot-inoculate Aesculin-bile Agar (A2.6.2) and incubate for 24 h for growth and blackening of the medium. Controls: positive - Enterococcus faecalis negative - Streptococcus agalactiae C3.4

Method 2 (Thornley, 1960). Stab-inoculate into Arginine Agar and pipette on to the surface a layer of molten sterile petrolatum (soft paraffin) to a depth of about 1 cm. Incubate at an appropriate temperature (pseudomonads at 30 °C, streptococci at 37 °C) and examine daily for up to 5 days. A positive reaction is shown by a colour change of the indicator to red. Controls: positive - Enterococcus faecalis negative - Streptococcus salivarius

Arginine hydrolysis

Method 1 Inoculate 5 ml Arginine Broth (A2.6.4) and after incubation for 24 h add 0.25 ml Nessler's reagent (Cl.ll). Arginine hydrolysis is indicated by the development of a brown colour. For streptococci, add 0.5 ml of culture to 4.5 ml distilled water, shake and add 0.25 ml Nessler's reagent.

C3.6

Bile tolerance

Inoculate onto 10% and 40% Bile Agar plates and incubate at 37 °C for 24-48 h. Growth indicates resistance to bile. Alternatively, 'ditch' plates comprising Blood Agar on one side and Bile Agar on the other may be used; strains should not be regarded as bile tolerant unless they grow right across the Bile Agar half. Controls: positive (10% and 40%- Enterococcus faecalis; Streptococcus agalactiae positive (10% only)- Streptococcus salivarius negative- Streptococcus dysgalactiae; Streptococcus pyogenes

223

APPENDIX C C3.7

The CAMP test (Christie, Atkins & Munch-Petersen, 1944)

Wash sheep erythrocytes with physiological saline to remove any antibodies to the CAMP' factor and resuspend the cells in saline to the original volume. Prepare CAMP plates by covering a layer of nutrient base (e.g. Oxoid Blood Agar No. 2) with a similar layer containing 10% of the washed sheep erythrocytes. Inoculate the plate with a single diametric streak of a p-toxin-producing strain of Staphylococcus aureus (NCTC 7428). Streak-inoculate the test strains at right-angles to but not touching the staphylococcal inoculum and incubate aerobically at 37 °C overnight. Examine the plates while still warm: diffusion of p-toxin will produce a zone of discolouration of the sheep erythrocytes. Production and diffusion of the CAMP factor from organisms under test will yield a completely clear area shaped like an arrow head in the zone of discolouration caused by the P-toxin. Alternatively inoculate the test culture to obtain discrete colonies. After incubation overnight, and with the plate still warm, place 0.02-0.04 ml (1 or 2 drops) of staphylococcal P-toxin (10 units/ml) on discrete colonies and re-incubate for a further 2 h before examining the plate. A clear zone of the medium around colonies covered by p-toxin is a positive CAMP test. Controls: positive - Streptococcus agalactiae negative - Enterococcus faecalis C3.8

Carbohydrate breakdown

Acetylmethylcarbinol production: see C3.1 C3.8.1

Acid from carbohydrates

Method 1. Peptone Water (A2.5.2) and Nutrient Broth sugars (A2.5.3). Inoculate and examine daily for 7 days for acid or acid and gas production. Reversion to alkalinity should also be noted. Negative tests should be examined at regular intervals for up to 30 days. With some organisms the ability to produce visible gas depends on the temperature of incubation, and if equivocal or suspect results are obtained the tests should be repeated at a lower temperature. As some anaerobes can produce gas from proteins, gas production is not reliable as an indicator of fermentation. When incubated anaerobically, some indicators may be 'bleached' (reduced to a colourless state); in such cases, test for acid production by the addition of fresh indicator solution. Method 2. Serum Water Sugars (A2.5.4). Inoculate and examine for acid production or acid and clot formation. Method 3. Media for neisserias and lactobacilli (A2. 5.6) 224

Inoculate and examine for growth and acid production. Method 4. Phenol-red Broth sugars for haemophili and streptococci (A2.5.3) Inoculate heavily with growth from Blood Agar medium, incubate at 37 °C and examine for acid production daily for up to 7 days (can normally be read after 1-2 days). For streptococci, use media containing 0.1% agar. Positive -Yellow Negative - Orange-red In case of equivocal results, check pH: a decrease to pH 5.0-6.3 indicates fermentation. Method 5. Blood Agar plate fermentation method for anaerobes (Phillips, 1976). Spread 1.0 ml of a sterile 20% solution of the fermentable substrate over the surface of a dried Blood Agar plate. Allow absorption of the solution to occur, redrying the plate if necessary. Spot-inoculate the test cultures onto the surface, and incubate anaerobically for 48 hours; a plain Blood Agar plate similarly inoculated should also be included as a negative control. To detect fermentation, remove a plug of the agar medium from the area of growth with a drinking straw, and expose it to a few drops of bromothymol blue indicator in a microtitre tray. Obtain and expose similarly negative control plugs both from the negative control plate and from an area of the test plate remote from bacterial growth. Use known positive cultures as controls to validate the test. To avoid false positive reactions in maltose fermentation tests, blood must be omitted from the agar fermentation base; it is conveniently replaced by haemin-menadione solution. Method 6. MRS Lactobacillus Agar for leuconostocs (A2.6.25). Despite heavy inocula, the production of gas from glucose by leuconostocs cannot always be detected in broth cultures by the traditional inverted inner (Durham) tube. Instead a few drops of a suspension from a fresh agar culture are mixed with 15 ml of melted MRS (glucose) agar in a test tube and cooled; when set, sterile Nutrient Agar is added on top to form a 'plug' about 1 cm in depth. After incubation for 1-2 days, leuconostocs produce sufficient gas to disrupt the MRS medium and force the plug upwards. Method 7. Ammonium Salt Sugars (ASS). Inoculate slopes of ASS medium (A2.5.5) and incubate at appropriate temperature for up to 7 days. Examine on alternate days for growth and acid production. P-galactosidase: see ONPG test (C3.42). C3.8.2

Oxidation or fermentation of glucose (Hugh & Leifson, 1953)

Some workers steam the OF medium to remove dissolved air, and quickly cool just before use. Stab-inoculate duplicate tubes with a straight wire. To

CHARACTERIZATION one of the tubes add a layer of melted soft paraffin (Petrolatum) to a depth of about 3 cm above the medium to seal it from air. Incubate and examine daily for up to 14 days. Results open tube sealed tube Oxidation yellow green Fermentation yellow yellow No action on blue or green carbohydrate green Note. This medium can also be used for detecting gas production and motility. Controls: oxidation - Acinetobacter calcoaceticus fermentation - Escherichia coli no action - Alcaligenes faecalis C3.8.3

Production of 3-ketolactose (Bernaerts & DeLey, 1963)

from

lactose

Inoculate a plate containing Medium 1 (A2.5.8) and incubate for 1-2 days; then inoculate a loopful of the resulting growth onto a plate of Medium 2 and incubate for 1-2 days. Flood this plate with a shallow layer of Benedict's reagent (C1.3) and leave at room temperature. Development around the growth of a yellow zone (of Cu2O) 1-2 cm in width within one hour indicates the production of 3-ketolactose. The yellow colour contrasts markedly with the blue reagent solution. Controls: positive - Agrobacterium tumefaciens negative - Pseudomonas aeruginosa C3.9

Carbon source utilization (CSU) tests

Apart from the citrate utilization tests (C3.9.1) and tests for use of organic acids (C3.9.2), other CSU tests are not yet well enough developed for use in identification. Those interested in their use in classification should consult Stanier, Palleroni & Doudoroff (1966) where the methods are described.

a slope of Simmons' citrate medium. Examine daily fpr up to 7 days for growth and colour change. Confirm positive results by subculture to Simmons' or Koser's Citrate medium. blue colour and streak of growth - citrate utilized original green colour - citrate not utilized Controls: positive - Klebsiella pneumoinae subsp. aerogenes negative - Escherichia coli Method 3. Inoculate Christensen's Citrate Agar by stabbing the butt and then drawing the wire over the surface of the slope. Examine for up to 7 days for colour change. magenta colour - citrate utilized yellow colour - citrate not utilized Note. An organism giving positive results in Koser's or Simmons' test will be positive in Christensen's medium, but an organism giving a positive result in Christensen's test may or may not be positive in Koser's or Simmons' citrate media. Controls: positive - Klebsiella pneumoniae subsp. aerogenes negative - Shigella sonnei C3.9.2

Inoculate the appropriate media and examine at intervals for up to 1 month; use of the carbon compound is indicated by alkali production, shown by colour change of the indicator from yellow to red Controls: Positive Negative Benzoate 1 Mucate \ Mycobacterium Mycobacterium Oxalate J smegmatis phlei C3.10

C3.9.1

Citrate utilization

Make a light suspension of the organisms in sterile water or saline; inoculate Citrate media with a straight wire. Incubate at 30 °C (enterobacteria) or optimum temperature (other organisms). Method 1. Inoculate Koser's citrate medium and examine daily for up to 7 days for turbidity. Confirm positive results by subculture again to Koser's citrate medium. Turbidity - citrate utilized No turbidity - citrate not utilized Method 2. Inoculate as a single streak over the surface of

Utilization of other organic acids as carbon sources

Catalase activity

Method 1. Incubate the organism on a slope of Nutrient Agar or other suitable medium; a supplement of 5% serum may be needed for fastidious organisms such as streptococci. Run 1 ml 3-6% H2O2 down the slope and examine immediately and after 5 min for evolution of gas, which indicates catalase activity. Method 2. Culture the organism in Nutrient Broth; after incubation overnight add 1 ml 3-6% H2O2 and examine immediately and after 5 min for evolution of gas. Notes. Blood Agar and other blood-containing media are unsuitable for the test. The addition of 1% glucose will avoid confusing reactions from pseudocatalase (Whittenbury, 1964).

225

APPENDIX C False positive results can be produced by dirty glassware; all tubes must be chemically clean (see Appendix Al.l). Cultures should be exposed to air for at least 30 min before adding H2O2 (Holdeman & Moore, 1972). Controls: positive - Staphylococcus saprophyticus negative - Enterococcus faecalis Method 3 for mycobacteria (Kubica, 1973). Inoculate the butts of Lowenstein-Jensen slopes enriched with 0.1 ml of 7-day cultures in Middlebrook 7H-9 broth (BBL or Difco powder with added ADC (albumin, dextrose (glucose) and catalase enrichment); incubate with caps loose for 2 weeks at 37 °C. Include with each batch of tests controls of a strong (Mycobacterium kansasii) and a weak (M. avium) catalase producer. Add 1 ml Tween 80-peroxide mixture (C1.19) to each culture and after 5 min measure the height of the column of bubbles above the surface of the medium. Results: negative - no froth (usually isoniazid-resistant strain of Mycobacterium tuberculosis or M. bovis) positive - froth < 45 mm (M. avium) hyperactive - froth > 45 mm (M. kansasii) C3.ll

Chitin hydrolysis (Holding & Collee, 1971)

Purified chitin (Lingappa & Lockwood, 1961) is made by treating crude chitin alternately with 1 N-NaOH and 1 NHC1 several times, and then with ethanol until foreign material is removed. Dissolve the purified chitin in cold cone. HC1, filter through glass wool, precipitate in distilled water, and wash until neutral. Add to melted Water Agar or Salt Agar at a concentration of about 0.25% (the medium should be slightly opaque) and pour as a thin layer on top of Nutrient Agar. Streak-inoculate the test organism(s) on the surface and incubate at the optimal growth temperature for the organism or at 22, 30 and 37 °C. Chitin hydrolysis is shown by a clear zone around the growth. Note. For halophilic organisms such as Vibrio parahaemolyticus the chitin should be suspended in Salt Agar, and the NaCl concentration of the basal medium increased to 2-3% or more. Controls: positive - Vibrio parahaemolyticus negative - Escherichia coli C3.12

Coagulase test

In tube tests, a positive result is indicated by definite clot formation; granular or ropy growth is regarded as doubtful and the organism should be retested.

226

Method 1 (Cowan, 1938Z?). Mix 0.5 ml undiluted plasma with an equal volume of an 18-24-hour broth culture and incubate at 37 °C for 4 h. Examine after 1 and 4 h for coagulation. If negative, the tubes should be left at room temperature overnight and then re-examined. Method 2 (Gillespie, 1943). To 0.5 ml of 1/10 dilution of plasma in saline add 0.1 ml of an 18-24-hour broth culture of the organism. Incubate at 37 °C and examine after 1, 3 and 6 h for coagulation. If negative, the tubes should be left at room temperature overnight and then re-examined. Method 3 (Cadness-Graves et al, 1943; Williams & Harper, 1946). With minimal spreading, emulsify a colony in a drop of water or saline on a microscope slide to produce a thick suspension. Stir the bacterial suspension with a straight wire which has been dipped into plasma. A positive result is indicated by macroscopic clumping within 5 seconds. Delayed clumping does not constitute a positive reaction. Method 4 (Subcommittee on taxonomy of staphylococci and micrococci, 1965). A single colony from a 24 h Blood Agar plate is emulsified in 1 ml fresh citrated rabbit plasma diluted 1 in 6 with physiological saline. Incubate in a water bath at 37 °C and read after 1, 2, 4, 8 and 14 h. If possible, include among the controls a weakly positive strain (e.g. NCTC 6571). This method detects free coagulase. Notes. If the plasma has been stored in a refrigerator it may be sufficiently cold to delay coagulation, especially in the slide test; it is advisable to allow the plasma to reach room temperature before use. The slide test is a valuable presumptive test but as it only detects bound coagulase, negative results should be confirmed in a tube test which will also detect free coagulase. Known positive and negative strains should always be tested in parallel and, with the tube tests, an uninoculated control should also be set up. Controls: positive - Staphylococcus aureus negative - Staphylococcus saprophyticus C3.13

Campylobacter tests for species differentation

C3.13.1 Hydrolysis of indoxyl acetate (Mills & Gherna, 1987) Add 50 jLil of a 10% (w/v) solution of indoxyl acetate in acetone to an absorbent paper disc 6mm in diameter and allow to dry in air. Apply growth from Campylobacter colonies directly to disc(s) and then wet with drop(s) of sterile distilled water. Appearance of a blue-green colour within 5-10 minutes indicates a positive result. Note. Dried discs are stable for at least 12 months if stored at 4 °C in a dark glass bottle with silica gel. Controls: positive - Campylobacter jejuni negative - Campylobacter fetus

CHARACTERIZATION C3.13.2 Rapid coccal transformation on exposure to air (Karmali, Allen & Fleming, 1981). Transformation to coccal morphology among campylobacters occurs on exposure to atmospheric oxygen and some species are much more prone to this change than others. To perform the test, incubate duplicate cultures on Mueller-Hinton or Blood Agar for 24 h under the microaerobic conditions normally used for campylobacters (5-10% O2); expose one plate to air and keep the other (control) plate under microaerobic conditions, both at ambient temperature. After 24-48 h compare the growth microscopically. Rapid coccal transformation (most of the cells coccoid) is a feature of the Campylobacter jejuni 'thermophilic' group, being most pronounced with C. lari and is and least with C. coli. Less than 5% of C. fetus cells are coccoid after such exposure to air. Helicobacter pylori forms cocci within one hour of exposure to air. Controls: positive - Campylobacter jejuni negative - Campylobacter fetus C3.14

Decarboxylase reactions

Method 1 (M0ller, 1955). From a plate culture, heavily inoculate, with a straight wire, tubes of the four media (arginine, lysine, ornithine, and control) through the paraffin layer. Alternatively, inoculate by adding a drop of a suspension of the organism above the paraffin layer and shaking to distribute the inoculum; the paraffin rises to the top on standing. Incubate and examine daily for up to 5 days. The media first become yellow due to acid production from the glucose; later, if decarboxylation occurs, the medium becomes purple. The control should remain yellow. With non-fermentative organisms, no acid (or insufficient acid) is produced from glucose and there is therefore no change of the indicator to yellow. Note. In the diagnostic tables, plus signs indicate only the production of a purple colour in the medium. When a positive reaction is obtained with arginine, the medium may be tested with Nessler' s reagent (C1.11) for the presence of NH3 in the absence of urease, the formation of NH3 indicates that the arginine dihydrolase system has taken part in the reaction (M0ller, 1955). Method 2 (Falkow, 1958). Inoculate tubes of the four media with a straight wire. Incubate and examine daily for up to 4 days. Decarboxylation is indicated by a purple colour, whereas the control and tubes with negative reactions are yellow. Falkow's method is not satisfactory with the genera Enterobacter and Klebsiella. Method 3 NCTC micromethod (Shaw & Clarke, 1955). Suspension: culture overnight on Nutrient Agar or other suitable medium, wash off in water, centrifuge and resus-

pend in a small volume of water so that the density is at least equal to 109 E. coli.ml. Test:

0.03 M-amino acid (pH 5.0) 0.0125 M-phthalate buffer, pH 5.0 with bromcresol purple Culture suspension

0.04 ml 0.04 ml 0.04 ml

Place the tubes in a 37 °C water bath. When decarboxylation takes place a colour change occurs; readings are taken after 2, 4 and 24 h. Each set of tests should include a suspension control without amino acid; when this control is alkaline (blue), the same volume of buffer required to render the control tube yellow should be added to all the amino acid tubes before reading them. This method is not suitable for decarboxylation of glutamic acid. Controls: Arginine Lysine Ornithine Proteus vulgaris + Aeromonas hydrophila + Klebsiella pneumoniae subsp aerogenes + Morganella morganii + + Salmonella typhi + + Enterobacter cloacae + + Enterobacter aerogenes + + + Salmonella typhimurium C3.15

Decomposition of tyrosine or xanthine

Inoculate plates of the appropriate media (A2.6.40; A2.6.42) and examine at intervals up to 5 days for dissolution of crystals under and around the bacterial growth. Controls: positive - Nocardia brasiliensis negative - Mycobacterium phlei C3.16

Digestion of casein

Inoculate plates of Casein Agar and at intervals examine for up to 14 days for clearing of the medium around the bacterial growth. Note. In a few instances clearing may be due to solution of the milk proteins by acid or alkaline metabolic products (see Hastings, 1903, 1904); this may be distinguished from true proteolysis by the addition of acid mercuric chloride (C1.2), when a decrease in the cleared area shows that the casein has not been digested. If soluble casein is used instead of skimmed milk, hydrolysis is detected by flooding the plate with acid mercuric chloride solution. Controls: positive - Bacillus subtilis negative - Mycobacterium phlei

227

APPENDIX C Note. For safety reasons, 30% trichloracetic acid may be used instead of acid mercuric chloride. C3.17

Digestion of egg

Inoculate a slope of Dorset egg and examine at intervals for up to 14 days for liquefaction. C3.18

Digestion of meat

Inoculate Cooked Meat medium and on the container mark the level of the meat. Incubate and examine at intervals for up to 14 days for diminution in the volume of the meat and for disintegration, or softening of the meat particles. Blackening or reddening of the meat and/or production of small white feathery crystals of tyrosine may also occur. C3.19

Digestion of inspissated serum

Inoculate a Loeffler serum slope and examine after incubation under appropriate conditions for liquefaction of the medium. C3.20

DNase (deoxyribonuclease) activity

Method 1 (Jeffries, Holtman & Guse, 1957). Dissolve DNA in distilled water; prepare sufficient to give a concentration of 2 mg/ml in the final medium. Add the DNA solution to Nutrient Agar (or other) base immediately before autoclaving and pour the plates as soon as the medium cools to 50 °C. Prepare replicate plates; inoculate test culture(s) as streaks on the surface. Incubate at several temperatures such as 25, 30 and 37 °C. After incubation for 36 h, flood the plates with 1 N-HC1; a positive result shows as a clear zone around the growth; the surrounding medium - and a negative result of the test - is opaque. Controls: positive - Serratia marcescens negative - Enterobacter aerogenes Method 2 (Lior & Patel, 1987). Spot-inoculate DNA Toluidine-blue Agar (A2.6.10) with a loopful of growth from a fresh Blood Agar culture and incubate microaerobically at 42-43 °C for up to 48 hours. A clear colourless or pink zone around the inoculum indicates the production of extracellular deoxyribonuclease. Controls: positive - Campylobacter jejuni biotype IV negative - Campy lobacter jejuni biotype I

228

C3.21

Eijkman test, modified 44 °C fermentation test (Report, 1983)

Inoculate MacConkey Broth (A2.5.9), warmed to 37 °C and incubate in a water bath at 44 ± 0.1 °C for 48 h. Regard the production of both acid and gas as a positive result. Note. Many alternatives to MacConkey Broth have been suggested, including the chemically defined Improved Formate Lactose Glutamate (IFLG) medium (Report, 1983). Controls: positive - Escherichia coli negative - Enterobacter cloacae C3.22

Ethylhydrocuprein (EHC) sensitivity

Place a disc impregnated with EHC (optochin, C 1.16) or a tablet containing 0.05 mg optochin on the surface of a Blood Agar plate inoculated with the organism. Incubate and examine after 18-24 h. Sensitivity to the compound is shown by inhibition of bacterial growth around the disc for a distance of at least 5 mm; resistant organisms will grow up to the disc or may show a small inhibition zone of not more than 1-2 mm. Controls: sensitive- Streptococcus pneumoniae resistant - Enterococcus faecalis Fluorescence; see A2.4.3 and A2.7.14. (3-galactosidase; see ONPG test (C3.39). C3.23

Gelatin hydrolysis (or liquefaction)

Method 1. Inoculate Nutrient Gelatin (A2.6.14) with a straight wire and incubate at 22 °C; observe daily up to 30 days for growth and presence of liquefaction. Method 2. Inoculate Nutrient Gelatin (A2.6.14) and incubate at 37 °C for up to 14 days; every 2-3 days, place in a refrigerator for 2 h and then examine for liquefaction. Set up a control tube of uninoculated medium in parallel. Method 3 (Frazier, 1926). Inoculate a slope or plate of Gelatin Agar (A2.6.14) and incubate for 3 days. Flood the surface with 5-10 ml acid mercuric chloride solution (C1.2); clear zones indicate areas of gelatin hydrolysis. When glass Petri dishes are used, metal tops should be avoided; plastic disposable dishes prevent contamination of glassware with mercuric ions. Note. 30% trichloracetic acid can be substituted for the acid mercuric chloride in Frazier's method (Pitt & Dey, 1970); for reasons of safety, this may be preferable. Method 4. (Ferrous Chloride Gelatin (A2.6.12) medium may be used for the determination of both gelatin liquefaction and H2S production (C3.30, method 2).

CHARACTERIZATION Method 5. Greene & Larks' (1955) adaptation of Kohn's (1953) gelatin discs to a micromethod, with a consequent increase in speed. Formalized gelatin discs are washed in water, sterilized by steaming for 30 min and stored in a refrigerator. Discs are added to 1 ml volumes of 1% Peptone Water, heavily inoculated with culture from an agar slope and placed in a 37 °C water bath. With positive cultures the discs disintegrate in 2-6 h. Controls: positive - Aeromonas hydrophila negative - Escherichia coli C3.24

Gluconate oxidation

Inoculate Gluconate Broth (A2.6.15) lightly and incubate at 37 °C for two days. Method 7. To 5 ml culture add 1 ml Benedict's qualitative solution (C1.3), mix, and boil for 10 min. The formation of a brown, orange, or tan precipitate constitutes a positive reaction. Method 2 (Carpenter, 1961). Add one Clinitest* tablet instead of the Benedict's reagent and look for the formation of a coloured precipitate as in method 1. Controls: positive - Klebsiella pneumoniae subsp. aerogenes negative - Escherichia coli C3.25

Growth on individual media

The ability to grow on a particular medium occurs in several tables. Media are inoculated and incubated for a suitable period in accordance with the growth rate of the bacteria. Growth should not be scored as absent until adequate time has elapsed for it to have occurred. The media on which this character is recorded in the tables are: Tables Acetate Agar 63a BCYE, BCYE (- CF) 7A0d Bile Agar (10%, 20% and 40%) 6.3(2,/?; 6.7; l.laM lAa Cetrimide media 7.5; 1.6a Chocolate Agar 7.106 Dye-containing Agar lAb Glycine 1% 7.10c MacConkey agar lAa\ 1.6a; 1.1; Media without salt

* Ames Division, Miles Laboratories Ltd, Stoke Poges, Slough SL2 4LY, UK.

Media with increased salt Nutrient Agar Selenite SSagar Triphenyltetrazolium chloride (TTC) 0.04% C3.26

63aM 6.5c; 6.9; lAa\ 7.8a,c; 7.10c 7.3; lAa 1.9a,b,c,d,e 7.5 7.10c

Growth in media with increased NaCl concentration

Inoculate broth of the required salt concentration with the organism to be tested and incubate at the optimal temperature for growth. Many salt-tolerant organisms (and most halophiles) have optimal temperatures below 37 °C For campylobacters, use Mueller-Hinton agar (1941) containing 1.5% NaCl. C3.27

Hippurate hydrolysis

Method 1 for streptococci. Culture the organism in Hippurate Broth (A2.6.17) for 2-4 days. Centrifuge the culture and, to 2 ml of the supernatant fluid, add 0.5 ml of a 50% solution of concentrated sulphuric acid. A fine white precipitate (of benzoic acid) indicates hydrolysis of the hippurate. Controls: positive - Streptococcus agalactiae negative - Streptococcus salivarius Method 2 (Thirst, 1957a). Lightly inoculate slopes of Hippurate Agar (A2.6.17) and examine daily for up to 7 days. Hydrolysis of hippurate is indicated by growth and a pink colour due to alkali production. Controls: positive - Klebsiella pneumoniae subsp. aerogenes negative - Enterobacter cloacae Method 3 (Hwang & Ederer, 1975). Suspend a loopful of growth from an 18-24 hour Blood Agar culture in 0.4 ml of a 1 % solution of sodium hippurate in phosphate buffer pH 7.0 (freshly prepared or stored frozen at -20 °C). Incubate at 37 °C for 2 hours with occasional mixing. Then slowly add 0.2 ml of a freshly prepared solution of 3.5% ninhydrin (C1.13) down the side of the tube to form an overlay. Reincubate at 37 °C for 10 minutes and examine for the development of a deep purple colour which indicates the presence of glycine, an end product of hippurate hydrolysis. Tubes showing a pale purple colour or remaining colourless indicate negative results. Controls: positive - Klebsiella pneumoniae subsp. aerogenes negative - Enterobacter cloacae

229

APPENDIX C Method 4 for Legionella pneumophila. Thaw the sodium hippurate solution (Section C1.16). Prepare a milky suspension of organisms from the growth (\^\ days) on BCYE medium (A2.3.6) and add a loopful to the hippurate solution. Incubate in air at 35 °C for 18-20 h. Add 0.2 ml of 3.5% ninhydrin solution (C1.13), mix gently, and reincubate at 35-37 °C for 10 min. Observe for development of a purple colour within 20 min. Regard a grey or light yellow colour as negative. Controls: positive - Legionella pneumophila negative - Enterobacter cloacae C3.28

Growth anaerobically with specific supplements

The ability of campylobacters to grow anaerobically on supplemented media is an aid to identification (Table 7.10c). The basal medium is semisolid Yeast Extract Agar to which the relevant compound(s) is added. Two or three stab inocula from a 24 h Blood Agar culture are made in the medium to about 1 cm below the surface. Incubate anaerobically and examine periodically for up to 7 days for growth throughout the medium. C3.29

Hydrogen peroxide production

This character is used in the differentiation of streptococcal species. Culture the organism in Infusion Broth (A2.1.6); centrifuge, discard most of the supernatant and resuspend the deposit in the remaining liquid. Immerse a commercial paper strip or disc containing peroxide reagent (C1.17) in the cell suspension: the rapid development of an intense blue colour indicates the presence of hydrogen peroxide. Controls: positive - Streptococcus pneumoniae negative - Streptococcus salivarius C3.30

Method 4 for Brucella species. Inoculate a Serum Glucose Agar slope (A2.2.6) and insert a lead acetate paper strip as in method 3. Examine and change the paper daily for 7 days, recording the results. Method 5 (Morse & Weaver, 1950). Medium: 2% thiopeptone in distilled water, pH 6.9; dispense in 0.8 ml volumes in 75 x 10 mm tubes; heat to 37 °C before heavy inoculation by means of a swab from a 6-hour culture on a solid medium. A 5 mm wide strip of lead acetate paper (C1.17) is folded 1 cm from the end and inserted into the mouth of the tube. Morse & Weaver say that the method is more sensitive than macrotests and that the 45 min reading gives the best correlation. Method 6 (Clarke, 1953a). Suspensions are made from growth on Nutrient Agar (see decarboxylase reactions (C3.14). 0.1 % cysteine hydrochloride, pH 7.4 0.04 ml 0.025 M-phosphate buffer, pH 6.8 0.04 ml Suspension of organism 0.04 ml Place a small lead acetate paper (C 1.17) in the mouth of the tube and keep it in position by a loose cotton-wool plug. Do not allow the paper to touch the suspension-reagent mixture. Incubate at 37 °C; read at intervals of 15 min for 1 h. Controls: positive - Proteus vulgaris negative - Shigella sonnei Method 7 (Rapid test of Skirrow & Benjamin, 1980, modified by Lior, 1984). A large loopful (5mm) of growth from an 18-24 h Blood Agar culture (incubated at 37 °C in not more than 7% O2) is gently suspended, without mixing, in the upper third of ?>-4 ml FBP medium (A2.6.11) in a small screw-capped tube or container. Incubate closed at 37 °C for 2 h. Blackening around the bacterial inoculum indicates a positive result. Controls: positive - Campylobacter jejuni (biotype IV) negative - Campylobacter jejuni (biotype I)

Hydrogen sulphide production

Method 1. Inoculate a tube of Triple Sugar Iron Agar (A2.6.38) by stabbing the butt and streaking the slope; observe daily for up to 7 days for blackening of the butt only due to H2S production. Some organisms produce a dark pigment on the slope only and this should not be mistaken for a positive result. Method 2. Stab-inoculate a tube of Ferrous Chloride Gelatin medium (A2.6.12) and incubate at 22 °C. Read daily for 7 days for blackening due to H2S production. Gelatin liquefaction may also be observed in this medium; for this incubation is continued for up to 30 days. Method 3. Inoculate the organism in Nutrient Broth or Peptone Water, and insert a lead acetate paper (C1.17) between the cap or plug and the tube. Examine daily for 7 days for blackening of the paper.

230

C3.31

Indole production

Method 1. Inoculate Peptone Water or Nutrient Broth, place an oxalic acid paper (C1.17) between the plug or cap and the tube. Incubate and examine daily for up to 7 days. Indole production is shown by the development of a pale pink colour at the lower end of the test paper. Method 2. Inoculate Peptone Water or Nutrient Broth and incubate for 48 h. Add 0.5 ml Kovacs' reagent for indole (C1.7), shake well, and examine after about 1 min. A red colour in the reagent layer indicates indole production. Method 3. To a 48-hour culture in Peptone Water or Nutrient Broth add about 1 ml ether or xylol; shake; run 0.5 ml Ehrlich's reagent (C1.5) down the side of the tube. A pink or red colour in the solvent indicates indole production.

CHARACTERIZATION Method 4 (Arnold & Weaver, 1948). Medium 1: Tryptone, 1%; meat extract, 0.3%, in distilled water. Medium 2: tryptophan, 0.03%; peptone, 0.1%; K2HPO4, 0.5% in distilled water. Adjust both media to pH 7.4. Inoculate medium heavily from a culture in the logarithmic phase; incubate in a 37 °C water bath. Test by adding 4 drops of Kovacs' reagent (C1.7), using iso-amy\ alcohol as solvent. Medium 2 gives positive results more quickly than Medium 1 (but with both media the tests should be complete within 2 hours). Method 5 NCTC micromethod (Clarke & Cowan, 1952). Suspensions are made from growth on Nutrient Agar (see Decarboxylase reactions, C3.14). 0.1% tryptophan 0.025 M-phosphate buffer, pH 6.8 Suspension of organism

0.06 ml 0.04 ml 0.04 ml

Incubate in water bath at 37 °C and test at 1 h by adding 0.06 ml Kovacs' reagent (C1.7) prepared with iso-amy\ alcohol; shake. Read immediately; red colour indicates indole production. Controls: positive - Escherichia coli negative - Enterobacter cloacae Indoxyl acetate hydrolysis test for differentation of campylobacters - see Campylobacter, C3.13.1 C3.32

KCN test

Inoculate 1 ml KCN Broth (A2.6.18) with one loopful of an overnight broth culture or a light suspension of the organism. Screw the cap of the bottle tight and incubate for up to 48 h. Examine after 24 and 48 h for turbidity indicating growth, which constitutes a positive reaction. Do not use a large inoculum. After use, care should be exercised in the disposal of cyanide-containing media; add a crystal of FeSO4 and 0.1 ml 40% KOH before sterilization. Controls: positive - Klebsiella pneumoniae subsp. aerogenes negative - Escherichia coli C3.33

incubator for 20 min. An insoluble bright greenish-blue copper soap is formed in those areas containing free fatty acid. Controls: opalescence - Clostridium perfringens pearly layer - Clostridium sporogenes no reaction - Bacillus subtilis C3.34

Malonate utilization

Method 1. Inoculate Malonate-phenylalanine medium (A2.6.22) lightly and incubate for 18-24 h. Examine for colour change and keep the culture for the phenylalanine deamination test (C3.45). A positive malonate reaction is indicated by a deep blue colour, a negative reaction by the unchanged greenish colour of the medium (with some negative strains a yellow colour appears). Method 2. NCTC micromethod (Shaw & Clarke, 1955). Suspensions are made from growth on Nutrient Agar (see Decarboxylase reactions, C3.14). 1% sodium malonate 0.025 M-phosphate buffer, pH 6.0 (phenol red) Suspension of organism

0.04 ml 0.04 ml 0.04 ml

Incubate in water bath at 37 °C; read at intervals up to 24 h. Red colour indicates a positive test. Controls: positive - Klebsiella pneumoniae subsp. pneumoniae negative - Proteus vulgaris C3.35

Methyl red (MR) reaction

Inoculate Glucose Phosphate (MR) medium (A2.10) and incubate at 30 °C for 5 days (some workers prefer 37 °C for 2 days). Add 2 drops of methyl red solution (C1.9), shake and examine. A positive MR reaction is shown by the appearance of a red colour at the surface. An orange or yellow colour should be regarded as negative. (After reading the MR reaction the same culture can be used for the VP test (C3.1).) Controls: positive - Escherichia coli negative - Enterobacter cloacae

Lecithovitellin (LV) reaction

Inoculate LV Agar (A2.6.21) and incubate for 5 days. Examine daily for (i) growth, (ii) opalescence within the medium under and around growth, (iii) 'pearly layer' formation over and around the colonies; (ii) and (iii) constitute positive reactions. Flood the plate with saturated aqueous CuSO4 solution, remove excess reagent and allow the plate to dry in an

C3.36

Milk, growth in (now rarely used diagnostically)

Inoculate the appropriate medium (A2.6.23) and examine daily for 14 days for colour change and clotting. The following reactions may be seen: Litmus milk: acid production indicated by pink colour, alkali production indicated by blue colour,

231

APPENDIX C reduction of the indicator shown by colourless (white) medium, acid clot shown by a firm pink clot which does not retract and which is soluble in alkali, rennet clot shown by a soft clot which retracts and expresses a clear greyish fluid (whey); the clot is insoluble in alkali; peptonization or digestion of the clot may follow, 'stormy fermentation' in which the acid clot is broken up by gas production. Purple milk: as above, but acid production is shown by a yellow colour. Reduction of the indicator is not seen. Ulrich milk: the colour changes may be interpreted as follows: bluish grey - unchanged, pale yellow green to yellowish orange - acid, pale bluish purple to reddish purple - alkaline, bluish green - oxidation, white - reduction, acidity or alkalinity are best observed in the lower (reduced) portion of the medium. Controls: acid - Escherichia coli alkaline - Alcaligenes faecalis stormy fermentation - Clostridium perfringens no change - Proteus vulgaris C3.37

For the enhancement of motility inoculate Craigie tubes (Craigie, 1931) or U-tubes containing semisolid nutrient agar; serial passages may be necessary. C3.38

Niacin test for mycobacteria

Method 1 (Marks & Trollope, 1960). Heavily inoculate the surface of Dorset Egg medium contained in a bijou bottle; add 0.4 ml sterile water and incubate with the cap loose for 2 weeks at 37 °C; a moist atmosphere must be maintained. Transfer the aqueous extract to a test tube and add 0.6 ml 4% ethanolic aniline followed by 0.6 ml 10% aqueous cyanogen bromide (C1.12). The presence of niacin is indicated by a yellow colour. Method 2 (Gutierrez-Vazquez, 1960; modified by Collins, Grange & Yates, 1985). Add 0.3 ml sterile water to a 30-day culture on Lowenstein-Jensen medium; autoclave at 115 °C for 15 min. When cool, remove 2 drops of liquor and place on a white tile. Add 2 drops 1.5% ethanolic o-toluidine and 2 drops 10% aqueous cyanogen bromide (C1.12). A positive result is shown by a pink to orange colour; negative reactions remain blue-grey. Note. Cyanogen bromide is toxic; the test must be carried out in a fume cupboard and, after completion, alkali should be added to decompose the reagent before sterilization. Controls: positive - M. tuberculosis negative- M.fortuitum

Motility

Method 1. Young broth cultures of the organism, incubated at or below the optimum growth temperature (e.g. 37 °C and 22 °C), should be examined in 'hanging drop' preparations, using a high-power dry objective and reduced illumination. Method 2. Stab-inoculate tubes of motility medium (A2.5.24) to a depth of about 5 mm. Incubate at or below the optimum growth temperature. Motile organisms migrate throughout the medium, which becomes turbid; growth of non-motile organisms is confined to the stab inoculum. Method 3 (for anaerobes). Take up some of the liquid from a young culture in Cooked Meat medium into the capillary of a Pasteur pipette; cut the capillary and seal both ends. A fine oxygen flame is needed to give a narrow zone of intense heat which melts the glass quickly. Examine the sealed capillary under the high-power dry objective. Notes. OF medium (A2.5.1) may be used to test motility but it cannot be relied upon to the exclusion of the methods described above.

232

C3.39

Nitrate reduction

Method 1. Inoculate Nitrate Broth (A2.6.26) lightly and incubate for up to 5 days. Note any gas formation in the inverted inner (Durham) tube. Add 1 ml of nitrite reagent A followed by 1 ml of reagent B (C1.1). A deep red colour which shows the presence of nitrite and thus shows that nitrate has been reduced, indicates a positive reaction. To tubes not showing a red colour within 5 min. add powdered zinc (up to 5 mg/ml of culture) and allow to stand. Red colour = nitrate present in the medium (i.e. not reduced by the organism). Absence of red colour = nitrate absent in the medium (i.e. reduced by the organism to nitrite, which in turn was itself reduced). Note. Incubation for 5 days is unnecessary with many organisms. Daubner (1962) reported that with the exception of two Erwinia species all members of the Enterobacteriaceae reduced nitrate to nitrite within 8 hours; it is convenient to test a sample of the inoculated medium daily and re-incubate if nitrate has not been reduced. Method 2. Brough (1950) used small volumes (1 ml in 75 x 10 mm tubes) of medium (0.1% KNO3 in Nutrient

CHARACTERIZATION Broth) warmed at 37 °C before inoculation with a heavy suspension and rapid return to the water bath at 37 °C. Test after 15 min with 3 drops each of Solution A and Solution B reagents (C 1.14). Method 3 Bachmann & Weaver (1951). Medium: peptone, 1%, meat extract, 0.3%; KNO3, 0.1% in distilled water; sterilize and store in flasks. Dispense in 1.0 ml volumes into clean, non-sterile 75 x 10 mm tubes. Heat tubes in 37 °C water bath and then inoculate with two 3 mm loopsful of 6-hour culture (inoculum should not be broken up but allowed to settle). After 15 minutes add 1 drop each of Solution A and Solution B reagents (C1.14). When nitrite is present a pink colour develops in 5 minutes. Method 4 NCTC micromethod (Clarke & Cowan, 1952). Suspensions are made from growth on Nutrient Agar (see Decarboxylase reactions, C3.14). 0.05% NaNO3 0.04 ml 0.025 M-phosphate buffer, pH 6.8 0.04 ml Suspension of organism 0.04 ml Incubate in water bath at 37 °C and test after 1 h. Add 0.06 ml nitrite test solution A and 0.06 ml test solution B (C1.14). Shake and read after 1-2 minutes. A pink colour is indicative of nitrite. In each series of tests a blank without suspension should be made to exclude the presence of nitrite in the substrate. Method 5 (Cook, 1950). Place a blotting paper strip (about 16 x 10 mm) containing potassium nitrate in the centre of a Blood Agar plate at the time of inoculation. The strips are prepared by soaking blotting paper in a warm solution of 40% potassium nitrate and then drying. Stabinoculate test and control organisms near the periphery of the plate and incubate for 24-48 h. A positive result is shown by a dark green-brown zone of methaemoglobin formed by the reduction of haemoglobin by nitrite. Controls: positive - Escherichia coli negative - Acinetobacter calcoaceticus C3.40

Nitrite reduction

Inoculate Nitrite Broth (A2.6.27) and incubate for 7-14 days. Add nitrite reagent Solutions A and B (C1.14) as for the nitrate reduction test. Red colour = nitrite present. Absence of red colour = nitrite absent, i.e. reduced by the organism. Controls: positive - Escherichia coli negative - Acinetobacter calcoaceticus C3.41

O/129 sensitivity

Method 1 (Davis & Park, 1962). Place a few crystals of

2,4-diamino-6,7-di-/.s0-propyl pteridine phosphate* on the surface of a plate previously inoculated with the test culture. Incubate the plate overnight and examine for inhibition of growth around the crystals. Method 2 (Bain & Shewan, 1968). Place an O/129 disc (C1.18) on the surface of a Nutrient Agar plate sown with the test organism. Incubate the plate overnight and look for a clear zone of inhibition around the disc. Method 3 (Furniss, Lee & Donovan, 1978). Place discs containing 10 fig and 150 L | Lg respectively of the pteridine reagent on the surface of medium previously inoculated with the test-culture. Note. These discs are available commercially. Controls: positive - Vibrio parahaemolyticus negative - Escherichia coli C3.42

ONPG Test (Lowe, 1962)

Inoculate a tube of ONPG broth (A2.6.28) and incubate for 48 hours; (3-galactosidase activity is indicated by the appearance of a yellow colour due to the production of o-nitrophenol. Controls: positive -Escherichia coli negative - Morganella morganii C3.43

Oxidase (cytochrome c oxidase) activity

Method 1 (Kovacs, 1956; modified by Cowan & Steel, 1974). A fresh solution of the reagent is prepared each time of use by adding a loopful of tetramethyl-p-phenylenediamine dihydrochloride to about 3 ml of sterile distilled water or saline; the reagent is not sufficiently stable for storage in aqueous solution even in the presence of ascorbic acid. However, solutions prepared with 0.1 % ascorbic acid can be kept at -20 °C and thawed when needed. For use, wet a filter paper disc in a sterile plastic Petri dish with a few drops of the indicator solution and smear the test culture (grown on a medium free from glucose and nitrate) across the moist paper with a platinum (not nichrome) loop, glass rod or swab stick. The appearance of a dark purple colour on the paper within 30 seconds denotes a positive reaction. Method 2 (Gaby & Hadley, 1957). Add 0.2 ml a-naphthol (C1.15, solution 2) and 0.3 ml 1% dimethyl-/?phenylenediamine oxalate (C1.15, solution 3) to an overnight broth culture. Shake vigorously. A blue colour appearing within 10 to 30 seconds indicates a positive result; weak reactions occurring between 2 and 5 minutes * Obtainable from BDH Chemicals Ltd., Broom Road, Poole, Dorset BH12 4NN, UK

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APPENDIX C should be ignored. For plate cultures mix equal volumes of the two reagents (C1.15, solutions 2 and 3) and run a few drops over the surfaces of colonies on the plate (Ewing & Johnson, 1960); colour changes and times are similar to the test in broth. Note. Method 2 is slightly less sensitive than method 1. Controls: positive - Pseudomonas aeruginosa negative - Escherichia coli C3.44

Oxygen preference of Mycobacteria

This may be determined by the character and rate of growth at the optimum temperature. Aerobes give 'mature' raised and confluent growth within one week on one or more slopes whereas microaerophiles either take considerably longer to attain mature growth or never do so. This differentiation can be determined more accurately with the semisolid medium of Marks (1976) and it is used to distinguish between M. tuberculosis and M. bovis. Liquid Medium base Na2HPO4 7.5 g KH2PO4 2.0 g 0.6 g MgSO4-7H2O Sodium citrate 2.5 g Ferric ammonium citrate 5 mg Tryptone 5.0 g Glycerol 20 ml Distilled water to 1000 ml Adjust pH to 7.3 and autoclave at 120 °C for 15 minutes. Semi-solid agar medium Add 1 g purified (ion) agar or similar to 1000 ml liquid medium base. Dissolve by steaming and then by autoclaving at 120 °C for 5 minutes. Shake well and dispense in 10 ml volumes in 14 ml screw-capped bottles and autoclave at 120 °C for 10 minutes. Before use add aseptically 0.5 ml calf serum. Inoculate with a vigorously growing culture and mix by inversion do not shake. Incubate at the appropriate temperature and inspect weekly. Aerobes grow in the top 1 cm and microaerophiles to a lower depth often giving a dense band of growth between 12 and 20 mm from the surface.

nine deaminase in Moraxella phenylpyruvica (Snell & Davey, 1971). Method 2 (Report, 1958). Inoculate heavily a Phenylalanine Agar slope (A2.6.30). Incubate overnight and run 0.2 ml of a 10% aqueous solution of FeCl3 over the growth. A positive result gives a green colour on the slope and in the free liquid at the base. Method 3 (Goldin & Glenn, 1962). Make a suspension of the test organism in a small tube. Place a phenylalanine test strip (C1.17) in the tube and let the tip of the paper touch the suspension. Place the tube in a water bath at 37 °C for 1 h, then add 1 drop of 8% aqueous FeCl3 solution to the strip. A positive test produces a green colour. Moraxella phenylpyruvica is not likely to give positive results unless incubated for more than 4 hours. Method 4 (Henriksen, 1950). Make a heavy suspension of the organism with the growth from one Nutrient Agar slope in 0.5 ml saline. Add 0.2 ml suspension to 0.2 ml 0.2% DL-phenylalanine in saline. Shake and place the tube in an almost horizontal position in an incubator. After 4 hours acidify the mixture by adding 10% H2SO4 using phenol red as indicator. Next add 4-5 drops of half-saturated aqueous FeSO4 (NH4)2SO4-6H2O, shake the tube and watch for a green colour (= positive) within a minute; the colour quickly fades. Method 5 NCTC micromethod (Shaw & Clarke, 1955). Suspensions are made from growth on Nutrient Agar (see Decarboxylase reactions, C3.14). 0.03 M-phenylalanine 0.025 M-phosphate buffer, pH 6.8 Suspension of organism

Incubate at 37 °C in a water bath. Test after 2 hours by adding 0.04 ml 2% aqueous ferric chloride; shake. Read a minute later; a green colour indicates the presence of phenylpyruvic acid. Controls: positive - Proteus vulgaris negative - Klebsiella pneumoniae subsp pneumoniae C3.46

C3.45

Phenylalanine deamination

Method 1 (Shaw & Clarke, 1955). After recording the result of the malonate test (C3.34) acidify with 0.1 N-HCI drop by drop until the medium is yellow. Then add 0.2 ml of a 10% aqueous solution of FeCl3; shake and observe any colour change. Watch immediately; a positive reaction is indicated by a dark green colour which quickly fades. Note. This method is not suitable for showing phenylala-

234

0.04 ml 0.04 ml 0.04 ml

Phosphatase test

Method 1. Lightly inoculate Phenolphthalein Phosphate Agar (A2.6.29) to obtain discrete colonies, and incubate for 18 hours. Place 0.1 ml ammonia solution (sp. gr. 0.880) in the lid of the Petri dish and invert the medium above it. Alternatively the medium may be exposed to ammonia vapour by holding above an open bottle. Free phenolphthalein liberated by phosphatase reacts with the ammonia and phosphatase-positive colonies become bright pink. Note. Baird-Parker (1963) recommends incubation of

CHARACTERIZATION cultures at 30 °C for 3-5 days before applying the test; this gives a greater number of positive results than in cultures incubated at 37 °C for 18 hours. Method 2 (White & Pickett, 1953). Substrate: dissolve 30 mg phenol-free phenyl disodium phosphate in 100 ml 0.01 M-S0rensen's citrate-NaOH buffer at pH 5.6 (keeps for 3 months at 4 °C in a stoppered bottle). Indicator: dissolve 50 mg 2:6-dibromo-Af-chloro-/?quinone imine in 10 ml methanol (stable for 2 months at 4 °C in a dark, stoppered bottle). Test: pipette 0.5 ml substrate into 100 x 13 mm tube. In this make a heavy suspension (1010/ml) of the organism under test; incubate the tube at 37 °C for 4 hours. Add 4 drops of indicator solution; shake and stand the tube at room temperature for 15 minutes. Add 0.3 ml «-butanol; shake, stand for 5 minutes. A purple or blue colour in the butanol layer indicates a positive result. Controls: positive - Staphylococcus aureus negative - Staphylococcus saprophyticus C3.47

Pigment production

For general purposes, inoculate a Nutrient Agar plate with a drop of a light suspension of the organism, incubate for 24 hours at 37 °C and then transfer to room temperature and observe for up to 5 days. Record the colours as red, orange, yellow, green, violet, brown, or negative i.e. produces none of these pigments. For Chromobacterium species use Mannitol Yeast Extract Agar (A2.4.2). Incubate at 22-30 °C. For mycobacteria, inoculate two Lowenstein-Jensen slopes directly from the primary isolate. Incubate one in continuous light; the other should be wrapped to exclude light or incubated in a closed box. Continuous light can be provided by placing a 'cold' fluorescent tube (e.g. 18" long) in the incubator (Collins, Lyne & Grange, 1989). For psychrophiles such as M. marinum exposure to day light at room temperature is acceptable. For most Pseudomonas species use King's media (A2.4.3). Incubate medium A at 37 °C for 24-96 hours and medium B at 37 °C for 24 hours followed by 22 °C (or room temperature) for 72 hours. Kligler's Iron Agar (KIA) and TSI enhance the pigmentation of Pseudomonas cepacia. Serratia marcescens strains vary in their media requirements for pigment production; try Nutrient Agar, Mannitol Yeast Extract Agar, or King's medium A at 22 °C or 30 °C. Note. Non-pigmented strains are not uncommon and a suitable medium for inducing pigment production by all recalcitrant strains has not yet been produced. Micrococci produce pigment well on potato, but staphylococci do not

seem to grow well enough on it to show any pigment. In general staphylococci produce pigment best in diffuse daylight. C3.48

Poly-p-hydroxybutyric acid (PHBA) accumulation

Accumulation of poly-p-hydroxybutyrate (PHB) can be seen by phase-contrast microscopy and confirmed by staining with Sudan black (Stanier, Palleroni & Doudoroff, 1966), or with dilute carbol fuchsin; it may be confirmed by chemical extraction (Williamson & Wilkinson, 1958). The methods are hardly routine in clinical laboratories, but if the information can be obtained it will be useful in identifying pseudomonads. Method: Inoculate Polyhydroxybutyrate Agar (A2.6.31) and examine after incubation for two days at the optimum temperature for growth. For the presence of PHB inclusions, examine films of the growth microscopically by staining with Sudan Black (B1.18). Dry the film in air and fix by flaming. Flood the slide with freshly filtered Sudan Black stain and leave at room temperature for 15 minutes. Drain off excess stain, blot and allow to dry. Rinse with xylol and blot dry. Counterstain lightly with 1% aqueous safranin for 5-10 seconds, rinse in running water and blot dry. A positive reaction is indicated by some but rarely all of the organisms showing a purple-black appearance under the microscope. C3.49

Polysaccharide formation on sucrose media

Inoculate Sucrose Broth (A2.5.3) and incubate. Much dextran formation is shown by an increased viscosity of the culture and the dextran may be flocculated by the addition of ethanol. The dextran is best recognized by a precipitin test in which the broth culture is set up against a capsular antiserum to Streptococcus pneumoniae type 2. Inoculate a plate of Sucrose Agar and incubate. Levanproducing organisms grow as large mucoid colonies; dextran-producing organisms (e.g. Streptococcus sanguis) may form small glassy colonies on this medium. Controls: dextran produced- Streptococcus sanguis levan produced - Streptococcus salivarius neither produced - Enterococcus faecalis C3.50

Porphyrin (ALA) test for haemophili (Kilian, 1974)

Enzyme substrate. Prepare 8-aminolaevulinic acid (ALA) 2 mM and MgSO4 0.8 mM in 0.1 M-phosphate buffer at pH 6.9 and filter-sterilize. The substrate can be stored in screw-

235

APPENDIX C capped glass bottles for several months at +4 °C and for use distribute in 0.5 ml volumes in small glass tubes as required. Method 1. Add a full loopful of bacterial growth from a 24-hour Chocolate Agar plate to the substrate and incubate for 4 hours at 37 °C. After incubation expose the tube to Wood's lamp (maximum emission 360 nm) in a darkened room. Red-fluorescence = positive for porphyrins, i.e. X-factor not required. Method 2. Set up the test as in method 1 and incubate for 24 h at 37 °C. Add 0.5 ml Kovacs' reagent (C1.7) and shake well. Red colour in lower aqueous phase = positive for porphyrins, i.e. X-factor not required.

Method 1. Flood the plate with Lugol's iodine solution (B1.12); the medium turns blue where starch has not been hydrolysed; clear colourless zones indicate hydrolysis. Method 2 (Kellerman & McBeth, 1912). Flood the plate with 95% ethanol; milky-white areas indicate no action on starch, whereas clear zones indicate hydrolysis. This reaction is not instantaneous and is best observed 30 minutes after addition of the ethanol. Note. Some strains of Bacillus species produce only restricted zones of hydrolysis that may not be obvious until the bacterial growth has been scraped away. Controls: positive - Bacillus subtilis, Streptococcus bovis, biotype 1 negative - Escherichia coli, Streptococcus salivarius

C3.51

C3.55

Pyruvate fermentation test for streptococci

Inoculate slopes of Pyruvate medium (A2.6.32) with test strains and incubate at 37 °C for at least 2 days and examine daily. The indicator changes from green to yellow if pyruvate is fermented. Controls: positive - Enterococcus faecalis negative - Streptococcus salivarius Rapid coccal transformation on exposure of air - see Campylobacter tests C3.13 C3.52

Salt tolerance of streptococci

Inoculate 4% and 6.5% Salt Broth (A2.6.33) containing 0.1% (w/v) glucose with the test organism and incubate at 37 °C for at least 2 days. Examine daily for growth. Controls: positive (6.5% NaCl)- Enterococcus faecalis positive (4% NaCl) - Streptococcus agalactiae negative - Streptococcus sanguis C3.53

Selenite reduction

Inoculate a tube of 0.4% Selenite Broth (A2.6.34) with a light suspension of the organism and incubate for 2 days. A positive result is indicated by the appearance of a brick-red precipitate of metallic selenium. Controls: positive - Salmonella subgenus HI negative - Buttiauxiella agrestis C3.54

Starch hydrolysis

Inoculate lightly Starch Agar (A2.5.13) or Nutrient Agar (A2.1.1) containing 0.2% soluble starch and incubate plates at 30 °C for 5 days. For streptococci, incubate at 37 °C in air with added CO2 for two days.

236

Streptococcal grouping

Preparation of the extract 1. Acid extraction (modified from Lancefield, 1933). Mix the growth from a quarter of a Blood Agar plate or the centrifuged deposit from a culture (10-25 ml) in Todd-Hewitt broth with 0.4 ml 0.2 N-HC1 and place in a boiling water bath for 10 minutes. Cool and neutralize the supernatant with 0.2 N-NaOH, using phenol red as the indicator. Centrifuge and use the supernatant. 2. Formamide extraction (Fuller, 1938). Mix the growth from a quarter of a Blood Agar plate or the centrifuged deposit from 5 ml of a culture in Todd-Hewitt broth with 0.1 ml formamide and heat in an oil bath at 160 °C for 15 minutes or until the growth is almost completely dissolved. Add 0.25 ml 95% ethanol containing 0.5% HC1, shake and centrifuge. Discard the precipitate and add 0.5 ml acetone to the supernatant. Shake, centrifuge, and discard the supernatant. Dissolve the precipitate in 0.4 ml saline, and neutralize with 0.2 N-NaOH using phenol red as the indicator. 3. Enzymic extraction (Maxted, 1948). Suspend a loopful of growth from a Blood Agar plate in 0.25 ml enzyme solution (C1.8) and heat in a water bath at 50 °C until clear (about 1-2 hours). The test method Prepare 'grouping pipettes' from 5 mm diam. glass tubing by drawing out one end into a capillary. Cut to a total length of about 3 cm and stand vertically by placing the capillary end in a block of Plasticine (modelling clay) or similar material. Place a drop of antiserum in the pipette and layer the extract on top. The formation of a precipitate at the interface of the two layers within 2-3 minutes indicates a positive reaction. Alternatively, use Ouchterlony-type double diffusion tests in agar on slides. Place the streptococcal extracts from the test and control strains and also the grouping antisera in

CHARACTERIZATION appropriate wells; incubate and observe for lines of identity between the test and control strains. Notes. While strains of groups A, B, C and G are those most often investigated in medical laboratories, the tests should be made against all available group antisera. Commercial kits are available for groups A, B, C, D, F & G. By Lancefield's method, some strains are better extracted with 0.07 N-HCI; others, particularly group B strains, yield better extracts by heating at 50 °C for 2 hours (Williams, 1958). Cross-reactions may occur when the extract prepared by the formamide method is too alkaline. This method tends to destroy the group O antigen. Maxted's enzyme method is suitable for streptococci of groups A, C and G but strains of other groups (especially D) are less easily lysed by the enzyme. Group D strains do not always give good reactions and, when suspected, should be grown in broth containing 0.5% glucose. C3.56

Streptococcal enzyme tests

Production of the enzymes leucine aminopeptidase and pyrrolydonylarylamidase (pyroglutamase or PYRA) are important characters for the identification and differentiation of streptococci. To detect their presence, prepare the respective substrates (L-leucyl-p-naphthylamide and L-pyrrolydonyl-P-naphthylamide) as 0.01% solutions in phosphate-buffered saline (pH 7.3) and sterilize by autoclaving at 121 °C for 10 minutes. For use add 200 jLil volumes of each substrate to separate small, capped tubes. Inoculate a tube of each substrate with growth from a fresh culture on Blood Agar to give a turbid suspension. Incubate at 37 °C for 4 hours and then add 50 JLLI of dimethylaminocinnamaldehyde in 10% (v/v) concentrated HC1 to each tube. A red colour developing within 30 seconds denotes a positive result. Controls: Leucine aminopeptidase positive - Streptococcus agalactiae negative - Staphylococcus saprophyticus Pyrrolydonyla rylamidase positive - Streptococcus pyogenes negative - Streptococcus agalactiae C3.57

String of pearls test (Charlton, 1980)

Bacillus anthracis produces swollen round cells in chains ('strings of pearls') when incubated for 3-6 hours on Tryptose Agar containing 0.05-0.5 units/ml of penicillin. To 100 ml of molten Nutrient Agar, add 1 ml of a solu-

tion of sodium benzyl penicillin containing 50 units/ml and mix carefully. Pour into Petri dishes and allow to set. With a scalpel cut a block about 1.6 cm2 from the penicillin-agar plate and place it on a microscope slide in a Petri dish containing a small piece of moistened absorbent cotton wool or filter paper to prevent the agar drying out. Use a young colony to streak inoculate the centre of the agar block. Place a clean coverslip marked on one side about 5 mm from one edge on the agar block so that the marked line is face down alongside the streak inoculum. The line acts as a focussing and location guide. With the lid on, incubate the Petri dish at 37 °C. After 2 hours, remove the slide and examine the inoculum microscopicaly by oil immersion for the 'strings of pearls' growth. C3.58

Tellurite tolerance

Inoculate Blood Tellurite Agar plates (A2.3.3) and incubate at 37 °C for 24 h. Tellurite-resistant organisms show a heavy growth of jet-black colonies; tellurite-sensitive organisms fail to grow or show a very light growth, the colonies usually being visible only with a hand lens. Control: positive - Enterococcus faecalis negative - Streptococcus agalactiae C3.59

Temperature range for growth

The general principle of this type of test is that a suitable medium is lightly inoculated with the organism under test and incubated at different temperatures. For temperatures above 37 °C, a thermostatically controlled (±0.5 °C) water bath is preferable to an incubator, though heating blocks are even better as they avoid the possibility of contamination from the water. With aerobic spore-forming bacilli, mycobacteria, and actinomycetes, a solid medium (as a slope) is preferable; with other organisms liquid media are used. For streptococci, Abd-el-Malek & Gibson (1948a) recommended Litmus Milk; for organisms showing little activity in plain milk they add 0.25% each of glucose and yeast extract. At 22 °C and above examine cultures for growth daily for up to 7 days; at lower temperatures after 7 and 14 days. The exact duration of incubation will depend upon the normal growth rate of the organism. The ability of an organism to grow at a particular temperature should be confirmed by growth at that temperature after subculture. C3.60

Temperature tolerance (60 °C) for enterococci and similar organisms

Place 1 ml of a 24-hour Nutrient Broth or Serum Broth cul-

237

APPENDIX C ture in a small test tube, and place in a water bath at 60 °C for 30 minutes. Cool under cold running water and incubate at 37 °C for 24 hours. Subculture to a Serum Agar slope and incubate for 24 hours. Examine for growth which indicates the ability of the organism to survive the temperature-time conditions. Controls: positive - Enterococcus faecalis negative - Streptococcus pyogenes C3.61

Tween 80 hydrolysis

Method 1 (Sierra 1957). Streak-inoculate the test culture on the surface of Tween 80 Nutrient Agar (A2.6.39); incubate at the optimal growth temperature of the organism. Look at the plate each day; an opaque halo of precipitation around the growth indicates hydrolysis of the Tween. Controls: positive - Pseudomonas aeruginosa negative - Bordetella bronchiseptica Method 2 (modified from Wayne, 1962). A solution of Tween 80 (0.5%) in phosphate buffer, pH 7.0, with neutral red as indicator, is distributed in 2 ml volumes in small screw-capped containers and autoclaved. The final colour should be amber. Store at 4 °C, protected from light, and use within 4 weeks. Inoculate with a large loopful of growth from Glycerol egg medium, incubate at 37 °C and read twice weekly for 3 weeks. Controls: positive - Mycobacterium kansasii negative - (i) uninoculated medium (ii) Mycobacterium fortuitum group C3.62

Urease activity

Method 1. Inoculate heavily a slope of Christensen's Urea medium (A2.6.41); examine after incubation for 4 hours and daily for 5 days. Red colour = positive. For Helicobacter pylori inoculate heavily 0.5 ml Christensen's Urea medium without agar. Shake well and examine for pink colour after standing for 5 minutes. Method 2. Inoculate heavily a tube of SSR Urea medium (A2.6.41); incubate and examine daily for 7 days. Red colour = urea hydrolysed. Method 3 (Stuart, van Stratum & Rustigian, 1945). Medium for rapid test: to 380 ml distilled water add KH2PO4, 364 mg; Na2HPO4, 380 mg; urea, 8 g; yeast extract, 40 mg; 0.02% phenol red, 20 ml; pH 6.8. Filter to sterilize. Test: pipette 1.5 ml volumes into small tubes and inoculate heavily with growth from a solid medium. Incubate at 37 °C in a water bath. Proteus species produce a red colour in 5-60 minutes.

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Method 4 (Hormaeche & Munilla, 1957). Test solution: to 100 ml 2% urea solution add 2 ml 0.04% cresol red. Distribute in 1 ml volumes into small tubes; sterilization is not necessary when the test is to be made on the same day. Inoculate the tubes heavily with growth from a 24-hour agar culture (older cultures are too alkaline). Place the tubes in a water bath at 45-50 °C for 2 hours. Urease-positive cultures produce a violet red colour. Method 5 for haemophili (Maslen, 1952). Inoculate Maslen's Urea Broth (A2.6.41) heavily with growth from a fresh agar culture not more than 18 hours old. Incubate at 37 °C for 5-18 hours. The rapid development of a red colour indicates urease activity. Method 6. NCTC micromethod. Suspensions are made from growth on either Nutrient or Glucose Agar; the latter usually give stronger reactions. 2% urea aqueous solution 0.025 M-phosphate buffer, pH 6.0 phenol red Suspension of organism

0.06 ml 0.06 ml 0.04 ml

Place in 37 °C water bath and observe at intervals of 15 minutes for the first hour; leave negative reactions for a final reading at 4 hours. Positive tests show a red colour. This is better than the test given by Clarke & Cowan (1952). Controls: positive - Proteus vulgaris negative - Escherichia coli Voges-Proskaiier (VP) reactions: see C3.1.

C3.63

'X' and ' V factor requirements

Method 1. Inoculate a Blood Agar plate and a Nutrient Agar plate with the organism and spot inoculate a strain of Staphylococcus aureus on each plate, Observe each plate for growth and for 'satellitism'. Growth on Blood Agar plate only = requires X factor Growth shows 'satellitism' on Blood Agar = requires X and V factors Shows 'satellitism' on both media = requires V factor Growth on both media but not = neither X nor V showing 'satellitism' factor required Method 2. Inoculate a Nutrient Agar plate and lay an Xfactor and a V-factor disc (C1.18) on the surface at a distance of about 2 cm from each other. Examine for growth in the vicinity of one or both discs.

APPENDIX D Test organisms

Most of the biochemical characterization tests described in this Manual should be controlled by organisms that are known to give either positive or negative reactions under appropriate conditions. For this purpose, we list in Table Dl the species recommended together with the accession numbers of suitable strains available from the National Collection of Type Cultures, Central Public Health Laboratory, Colindale Avenue, London NW9 5HT and, in many instances, also from the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA. As controls for the common tests shown in Table D2, the set of four strains given is adequate. For other bacterial characters, the test strains shown in Table D3 may be used. We emphasize, however, that most of the organisms shown in these tables are pathogenic so that due regard must be paid to safety and hygiene in the laboratory. For the maintenance and storage of test-control strains, freeze-dried (lyophilized) cultures which retain their characters and keep virtually indefinitely are ideal. However, for those who wish to keep frequently used strains for immediate use, we give in Table D4 suggestions for the nutrient media, cultural conditions and short-term storage period in order to be reasonably sure of their survival before they need replacement from stock cultures. Table Dl. Recommended strains of test organisms

Acinetobacter calcoaceticus Aeromonas hydrophila A Icaligenes faecalis Bacillus stearothermophilus Bacillus subtilis Bordetella bronchiseptica Buttiauxella agrestis Campylobacter jejuni biotype I Campylobacter jejuni biotype IV Campylobacter fetus subsp. fetus Clostridium perfringens Clostridium sporogenes Clostridium tetani (toxigenic) Clostridium tetani (non-toxigenic strain)

NCTC

ATCC

7844 7810 655 10003 3610 452 12119 11168 12145 10842 6719 533 279 6336

15308 9071

Enterobacter aerogenes Enterobacter cloacae Enterococcus faecalis Escherichia coli Helicobacter pylori Klebsiella pneumonial subsp. aerogenes Legionella pneumophila Morganella morganii Mycobacterium tuberculosis Mycobacterium fortuitum Mycobacterium kansasii Mycobacterium phlei Mycobacterium smegmatis Nocardia brasiliensis Nocardia otitidis-caviarum Proteus vulgaris Pseudomonas aeruginosa Salmonella typhi Salmonella typhimurium Serratia marcescens Shigella dysenteriae 1 Shigella sonnei Staphylococcus aureus Staphylococcus saprophyticus Streptococcus agalactiae Streptococcus anginosus ('S. milleri') Streptococcus dysgalactiae Streptococcus pneumoniae Streptococcus salivarius Streptococcus sanguis Vibrio alginolyticus Vibrio furnissii Vibrio parahaemolyticus

NCTC

ATCC

10006 10005 8213* 9001 11637 418 11192 10041 *> 7416 10394 10268 8151 8159 10300 1934 4175 7244 786 74 1377 4837 8220 8532 4276c 8181 10708 4669 7465 8618^ 7863* 12160 11218/ 10903

13048 13047 11775 15380 33152 9360 6841 14471 19249 19420 19295 14629 13315 7700 13311 274 13313 12600 13813 10015 7073 10556 17749 17802

a

6051 19395

9856 10000 19406

Listed as Streptococcus faecalis in NCTC Catalogue (1972, 1989). b Listed as Proteus morganii in NCTC Catalogue (1972, 1989). c Listed as Micrococcus sp. subgroup 1 in NCTC Catalogue (1972), and as Staphylococcus epidermidis in Cowan & Steel (1974). d Listed as Streptococcus hominis in NCTC Catalogue (1972) and as Streptococcus species Group K in NCTC Catalogue (1989). e Listed as Streptoccus species Group H in NCTC catalogue (1972). / Listed as Vibrio fluvialis in NCTC Catalogue (1989).

239

APPENDIX D Table D2. Control strains for commonly used tests NCTC Strain Positive Negative Aesculin hydrolysis Citrate utilization Decarboxylases Arginine Lysine Ornithine Deoxyribonuclease Gelatin liquefaction Gluconate oxidation Hydrogen sulphide (TSI) Indole production KCN tolerance Malonate utilization Methyl red ONPG PPA production Selenite reduction Urease NCTC NCTC NCTC NCTC

11935 11934 7475 11936

= Serratia marcescens = Edwardsiella tarda = Proteus rettgeri = Enterobacter cloacae

240

11935 7475

11934 11934

11936 11935 11935 11935 11935 11936 11934 7475 7475 11936 7475 11935 7475 11936 7475

7475 7475 7475 11934 11936 7475 7475 11935 11934 7475 11935 7475 11936 11934 11935

Test Voges Proskaiier Gas from glucose Acid from: Adonitol Arabinose Dulcitol Inositol Lactose Maltose Mannitol Raffinose Rhamnose Salicin Sorbitol Sucrose Trehalose Xylose

NCTC Strain Positive Negative 11935 11936

7475 11935

7475 11936 11936 11935 11936 11936 11936 11936 11936 11935 11936 11936 11936 11936

11934 11934 11934 11934 11934 7475 11934 11934 11934 11934 11934 11934 11934 11934

TEST ORGANISMS Table D3. Further tests and recommended control strains Test

Control organism

Bacitracin sensitivity

Streptococcus pyogenes Streptococcus viridans Staphylococcus aureus Streptococcus pyogenes Staphylococcus aureus Staphylococcus saprophyticus Staphylococcus aureus Staphylococcus saprophyticus Streptococcus pyogenes Streptococcus viridans Staphylococcus saprophyticus Pseudomonas aeruginosa Serratia marcescens Acinetobacter Iwoffii Serratia marcescens Acinetobacter Iwoffii Serratia marcescens Acinetobacter Iwoffii Streptococcus pneumoniae Streptococcus viridans Pseudomonas aeruginosa Acinetobacter Iwoffii Staphylococcus aureus Staphylococcus saprophyticus

8198 10712* 8532 8198 8532 4276 8532 4276 8198 10712* 4276 10662 11935 5866 11935 5866 11935 5866 10319 10712* 10662 5866 8532 4276

Corynebacterium diphtheriae Corynebacterium diphtheriae Corynebacterium diphtheriae Haemophilus influenzae Haemophilus parainfluenzae Haemophilus canis

10648 3984 10356 10479 10665 8540

Catalase Coagulase Deoxyribonuclease Haemolysis Oxidation/Fermentation Motility Nitrate reduction Optochin sensitivity Oxidase Phosphatase Toxigenicity testing of C. diphtheriae X- and V-factor

•

NCTC number

ATCC number

Expected result

12344

Sensitive Resistant Positive Negative Positive Negative Positive Negative (3-haemolysis a-haemolysis No haemolysis Oxidative Fermentative Alkaline or negative Motile Non-motile Positive Negative Sensitive Resistant Positive Negative Positive Negative

12600 12344 12600 12600 12344 25668 15309 15309 25668 15309 15309 12600

19409

Positive Weak positive Negative Requires X and V Requires V Requires X

* Listed in NCTC Catalogue (1989) as Streptoccus species 'viridans' type; probably S. mitior.

241

APPENDIX D Table D4. Suggested conditions for subculture and short-term storage of test organisms Incubation

Genus

Medium

Acinetobacter Aeromonas Alcaligenes Bacillus Clostridium Enterobacter Escherichia Klebsiella Mycobacterium^ Neisseria Nocardia Pseudomonas Salmonella Shigella Staphylococcus Streptococcus Enterococcus Vibrio Vibrio (marine)

Nutrient agar Nutrient agar Nutrient agar Nutrient agar Cooked meat Nutrient agar Nutrient agar Nutrient agar Lowenstein- Jensen Chocolate agar Yeast extract glucose agar Peptone water agar Dorset egg Nutrient agar Nutrient agar Cooked meat or Blood broth Sloppy agar Salt-water agar or 3% NaCl agar

Temp. (°C)

Time (hours)

Storage*

37 37 37 30 37 37 37 37 37 37 37 30 37 37 37 37 37 30 30

18 18 18 18 18 18 18 18 48-72f 18 48-72 18 18 18 18 18 18 48 48

5-25 5-25 5-25 5-25 5-25 5-25 5-25 5-25 15-25 25-35 15-25 5-25 5-25 5-25 5-25 5-25 5 22-25 15-20

Maximum storage period (months) 3 3 3 12 12 6 6 3 3 7days 3 3 12 6 3 3 1 1 1

* In the dark. t Mycobacterium tuberculosis and other slow-growing mammalian pathogens should be incubated for 10-20 days or until growth is apparent to the naked eye.

242

APPENDIX E Preparation and use of Identicards

El

Identicard set for genus identification

Use a card with a single row of holes (Fig. 9.1). For the master cards, the key to the numbered holes for the characters is shown in Table E l , and the plan for notching the holes in Table E2. All positive characters are notched from the hole to the edge of the card. For certain genera two or more cards may be needed to make provision for different reactions ('dee' characters) or differences in reading a test (e.g. catalase with Aerococcus). A guide card with the holes numbered or labelled is useful for the probing (sorting) operation. Notching the unknown. Positive characters of the strain under test are notched from the appropriate hole (Table El) to the edge of the card. Probing. When notching the card for the unknown strain is complete put it at the front of the pack of master cards, correctly faced, and test each hole in turn with a probe such as a knitting needle. A probe through the hole of a positive character of the unknown strain will, when lifted, remove master cards that are negative for that character and these can be discounted. The card for the unknown strain and master cards of genera in which that character is positive will drop out of the pack; these cards are collected together and tested again for another character, and so on. Conversely, when unnotched holes (negative characters) of the unknown strain are probed, the cards that are lifted out with the probe are retained for further testing, and those that drop out are discounted (Table E3). The identity of the unknown strain (as far as can be determined by the set of cards used) will correspond with that of the master card that has similar notchings. E2

Identicard set for species identification

Use cards with two rows of holes (Fig. 92a). In the master cards for species, the outer row of holes is notched for characters recorded as d, (d) and (+) in the tables in Chapters 6 and 7. Characters shown as + in the tables are

notched to the inner row of holes (Fig. 9.2b). As an example, the notching plan for the enterobacteria is given in Tables E4 and E5. Table El. Key for numbered or labelled holes in the Identicard set for Genera (card with single row of holes) Hole no. 1 2 3 4 5 6 7 8 9 10 11 12

Character represented Gram reaction Motility Acid fastness Spore formation Coccus (bacillus [rod] is not notched) Growth anaerobically Growth in air Oxidase Catalase Optional Oxidative attack on sugars Fermentative attack on sugars

Notching the unknown. Positive characters of the unknown organism are notched to the second (inner) row of holes in the two-row cards (Fig. 9.2c). Probing. The completed card(s) is placed in front of the pack of master cards, correctly faced, and the probe inserted through the appropriate holes. In species identification, the probing procedure is different from that for genus identification (see Table E6). Positive characters of the unknown strain are probed in the first (outer) hole, and the probe lifted to remove master cards in which that character is negative. The cards that drop out are retained for probing other characters in sequence on the card. Negative characters are probed through the second (inner row) hole, and the cards that are retained on the probe are kept for further tests. As with the cards for generic identification, the probing is continued until only one master card remains or all the holes have been probed.

243

APPENDIX E

Table E2. Notching of master cards for Identicard set for genera Hole no.

1

Aerococcus (a) Aerococcus (b) Aeromonas Aeromonas salmonicida Achromobacter Acidaminococcus Acinetobacter (a) Acinetobacter (b) Actinobacillus Actinomyces Agrobacterium Alcaligenes Anaerobiospirillum Arachnia

V V

Arcanobacterium Arcobacter Bacillus (a) Bacillus (b) Bacillus (c)

V

Bacteroides (a) Bacteroides (b) Bacteroides (c) Bifidobacterium Bordetella Branhamella Brochothrix Brucella Campylobacter (a) Campylobacter (b) Cardiobacterium Chromobacterium Clostridium (a) Clostridium (b) (asporogenous) Corynebacterium Eikenella (a) Eikenella (b) Enterobacteria (a) Enterobacteria (b) Enterobacteria (c) Enterococcus Erysipelothrix Eubacterium Elavobacterium Francisella Fusobacterium Gardnerella Gemella (a) Gemella (b) Haemophilus (a) Haemophilus (b) Helicobacter Janthinobacterium Kingella

244

2

3

4

V V

V

V

6

7

V V

V V V V

V V V V V

V V

V V V

V V V

V V V V V V

V

5

V V V V V V V

V V

V

V

V

V V V V V V

V

V V V

V V V

9

V V V

V V V V

V V V

V

V V V

V V

V V

V V

V V

V

V

V

V V V

V V

V V

V V V

V V V V V V V

V V V V V V V

V V V V V V V V V

V V

V V

V V

Coccobacillus - coccus Coccobacillus - bacillus Gas not produced May branch

V

V V V V

V

Catalase = Catalase = w Mesophilic Psychrophilic (37 °C = no growth). Brown pigment.

V V

V V V V V V V

V V V V V

V

V V V V

V

V

V

V V

V V V

V V V

V V V V V V V V

V V

V V

V V V V V V

V

V

V V V

V V V V

V

V V V V

Examples of notes for centre of cards

V V V V

V

V V V V

V

10 11 12

8

V V

V V

V V

(Also Actinomyces odontolyticus) Branched, filamentous Microaerophilic Curved rods; 3-10% 02 Oxidase variable, ?inconstant Some spp. facultative [0] Carbohydrates: some spp. F some spp. O some spp. One sp. (B. anthracis) non-motile Catalase variable Most spp. non-motile One sp. motile Tendency to branch Does not grow on simple media Catalase at RT Carboxyphilic; urease + Will not grow under strict [0]; optimum = 5-10% O2. Catalase = D Pleomorphic; metachromatic granules? Mesophilic; growth at 37 °C. Violet pigment C. perfringens; non-motile; seldom spores Pleomorphic Needs CO2 for isolation; soon [O] probably cannot grow [0]; ? other anaerobic species Most produce gas; NO3 reduced Non-motile spp. Shigella dysenteriae 1 (Shiga's bacillus) Faecal streptococci Yellow pigment Enriched media required; dangerous Catalase variable Fastidious Easily decolorized; usually regarded as Gram-negative Fastidious; needs X and/or V factor; catalase variable Curved spiral rods; capnophilic; O2 6% Psychrophilic (37 °C = no growth). Violet pigment

IDENTICARDS

Table E2. (contd.) 2

Hole no.

1

Kurthia Lactobacillus Legionella (a) Legionella (b) Leuconostoc Listeria Megasphaera Micrococcus Mobiluncus Moraxella Mycobacterium Neisseria Nocardia (a) Nocardia (b) Ochrobactrum Pasteurella Pediococcus Peptococcus Peptostreptococcus Plesiomonas Prop ion ibacte Hum Pseudomonas Rothia

V V V

Shewanella Staphylococcus Stomatococcus Streptobacillus * Streptococcus Veillonella (a) Veillonella (b) Vibrio

3

V

V

V

V V

V

V V V V V V V

V

V V V

V

V = notched (positive character)

V

5

6

V V V V V V V V V V

V V V V

4

7

8

9

V V V V V V V V

V

V

V

V V V V V V V V V V V V V V V V V V V V V

V V

V V V V V V

V V V V V V V

10 11 12

V V V V V V V V

V V V V V V

V V V V V V V

V V V V V V V V V V

V V

V V V V V V

V

V V V V

V V V V

V

Examples of notes for centre of cards Can grow at low temperatures Grows best at about pH 6 f Requires cysteine and iron for growth; catalase + I but some strains give weak reaction May form dextran Can grow at low temperatures Large cell size (< 2 Jim) Colonies often pigmented Curved anaerobic rods Growth improved with blood or serum Does not branch or produce aerial hyphae Prefer added CO2 Feebly acid-fast; produce aerial hyphae Some spp. (strains) not acid-fast Achromobacter group A Gas not produced Tetrads common Metronidazole-sensitive Gas may be produced; metronidazole-sensitive Gas not produced; indole +; gelatin Club shapes; branching May produce fluorescent or green pigment f Filamentous; no mycelium or hyphae. Will 1 grow under microaerophilic conditions Pink pigmentation; copious H2S Needs enrichment; sensitive to Liquoid f Catalase variable I Catalase variable Gas not produced. Arginine -; lysine +; ornithine +

* Also Shigella dysenteriae 1

See Table El for key to holes. See Tables 5.1 and 5.2 on p.47 for explanation of the symbols used in the column of Notes.

245

APPENDIX E Table E3. Notching and probing of cards (single row) and action after probing Cards from probed set Character of unknown strain

Card of unknown strain

Positive

Negative

246

remaining on probe

dropping out

Notched

Discount (ignore)

Keep and retest for other characters

Not notched

Keep and retest for other characters

Discount (ignore)

Table E4. Key to numbered holes for species Identicard set (card with two rows of holes) for enterobacteria Hole No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Character represented Growth in KCN medium Utilization of citrate as C source Gas from glucose VP reaction Gelatin hydrolysis Urease Phenylalanine deaminase (PPA test) Motility H2S production from TSI Malonate Arginine dihydrolase Lysine decarboxylase Ornithine decarboxylase Optional Optional Adonitol, acid Dulcitol, acid Mannitol, acid Lactose, acid Indole produced

IDENTICARDS Table E5. Notching of master cards for species Identicard set (card with two rows of holes) for enterobacteria encountered clinically Hole No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 .17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46

Escherichia coli Escherichia adecarboxylata Edwardsiella tarda Yersinia pestis Yersinia pseudotuberculosis Yersinia enterocolitica Shigella dysenteriae 1 Shigella dysenteriae 2-10 Shigella flexneri 1-5 Shigella flexneri 6 Shigella boydii Shigella sonnei Citrobacterfreundii Citrobacter koseri Cedecea spp. Salmonella typhi Salmonella pullorum Salmonella gallinarum Salmonella choleraesuis Salmonella Subgenus I Salmonella Subgenus II Salmonella Subgenus III Salmonella Subgenus IV Erwinia herbicola Morganella morganii Proteus vulgaris Proteus mirabilis Proteus penneri Providencia rettgeri Providencia alcalifaciens Providencia stuartii Hafnia alvei Serratia marcescens Serratia liquefaciens Serratia marinorubra Serratia plymuthica Serratia odorifera Enterobacter cloacae Enterobacter aerogenes Klebsiella oxytoca Klebsiella pneumoniae subsp. aerogenes Klebsiella pneumoniae subsp. pneumoniae Klebsiella pneumoniae subsp. ozaenae Klebsiella pneumoniae subsp. rhinoscleromatis Kluyvera spp. Tatumella ptyseos

1

2

3

5

4

V V V

V

6

7

8

9

10 11 12 13 14 15 16 17 18 19 20

o

o

V V V V

V V

V V

o o o

V V V V V V V V o V o V V V V V V V V V V V V V V o

o

V V V V V V V V V V

o

V V V o V V V o V

o o o o V V V V V

V V V V V V

V V V V V

V o o V V V V

V = Notched to the inner row (plus line): usually positive Blank (no entry) = Negative

V V V V

V V V V V V V V V V V V o V o V o V V V V V V V V V V V V V V V V o V V V V V V V V V V V V o V V o V V V V V o o V o o V V o V V

o

V

V V

o o o o V o

V

V

V

o V V V V V V V V V V V o o

V

V

o V o

V V

V V V

o o o

o

o

V

o

o V V V V V O V o

o

V V V V V V V V

V V V V V V

V

V V V V V V

V V

o o o o

V V V V V V

V V V V o V V V V V V V V

o o o

o

V

V V

o o

V V

o o

o o

V

V

V V V

o

V V

V V V V V V

V V

o

V

o

o

V

o V

V

o o o V o

V V V o V V

o

V V V V V V V o V V V V V o V V V V V

O = Notched to the outer row (dee line): sometimes positive

See Table £4 for key to holes.

247

V

o

V

o

o o o o

o o

V V V V V V

V V V V V

o o

APPENDIX E

Table E6. Notching and probing of cards (double row) and action after probing Cards from probed set

248

Character of unknown strain

Card of unknown strain

Row tested

Remaining on probe

Dropping out

Positive

Notched to inner row

Outer

Discount (Ignore)

Keep for retest

Negative

Not notched

Inner

Keep for retest

Discount (Ignore)

APPENDIX F Use of computers

The availability of laboratory-based computers is increasing considerably, not only because of reductions in their price and size but also because programs suitable for epidemiological and microbiological purposes are more readily available. Historically, laboratory computing developed from the work of relatively few laboratories with limited access to the large mainframe computer facilities available in the 1960s. These were the 'first generation' of laboratory computers. They have all been replaced now by second and even third generation computer 'hardware' (the collective term used to describe the computer processor, the magnetic disk or tape storage apparatus as well as the visual display terminals and printer) and the associated 'software' (the programs for various applications). With the development of the 'silicon chip' all the associated hardware and software is available now for small sized micro-computers which have memory power and data storage capacity equivalent to those of their large predecessors. Any detailed description will undoubtedly soon be out of date but computer facilities with visual display terminals, printers, and adequate magnetic disk storage for as much as three years' clinical laboratory workload together with the necessary software (and training) are currently available at a modest cost. Such computer systems are well suited not only for bacterial identification purposes but also for a broad range of other uses in the microbiology laboratory. For those considering the use of computers but without experience of them, we give here brief guidance on how to go about it and list the basic essentials necessary for bacterial identification purposes. As a useful general introduction to the wide variety of computer equipment and applications currently available, 'Computers and their Use' by Carter & Huzman (1984) is recommended. Like practical bacteriology, however, there is no substitute for experience. It is therefore important to appreciate that the value of computers depends not only on the programs used to instruct them (and thus on the skills of programmers) but also on the quality of the data used; hence the aphorism 'garbage in, garbage out'. A complete 'computer package' consists of the actual

computer processor - the electronic 'brain' - which is designed to receive and act upon instructions conveyed to it through a keyboard. These instructions and the ensuing results are displayed visually either in colour or in monochrome on a video display unit (VDU) or monitor screen. The means or 'language' by which the computer is instructed or programmed via the keyboard varies according to its use and function. The usual 'languages' used with microcomputers include BASIC (Beginners All-Purpose Symbolic Instruction Code), ' C , PASCAL (named after a French scientist), SQL (Structured Query Language), MUMPS (Massacchusetts General Hospital Utility MultiProgramming System), FORTRAN (Formula Translation) and COBOL (Common Business Oriented Language). The various languages available simply mean that different keyboard letters or symbols and different sequences are used to represent specific programming instructions for different computers, many of which are designed for particular functions and objectives. Most of the languages mentioned can be used with many different computers though sometimes a particular model can provide a specific function which might not be available with other computers. Although understanding how to use the keyboard language to instruct the computer is not easy for the inexperienced, all software is supplied with complete and detailed instructions for keyboard use. The American National Standards Institute (ANSI) has promoted standardization of some of these computer languages by providing and updating guidelines for their compliance and use. The 'standardized' languages accepted by ANSI include BASIC, FORTRAN, COBOL, MUMPS and SQL. Provided that written programs keep to the ANSI recommendations, they should be capable of ready conversion so as to be compatible for use with a wide range of computers. Each language has its own particular advantages and disadvantages but they all provide the means for instructing the computer. The languages cited are termed 'high level' because although they use clear English-type keyboard terms such as INPUT, ENTER, PRINT, etc, these instructions have to be 'interpreted' and converted into 'low level' binary code language (termed

249

APPENDIX F machine code) either built into the computer or by using a software program called a 'compiler'; similarly, an 'assembler' program may be required to convert 'low level' language into machine code. Low level languages are usually much faster in use as the computer processor has less interpretation to do. However, writing such programs tends to be time consuming because the instructions are not in normal everyday language. Conversely high level languages are usually faster to instruct but slower for the computer to execute. All these and other factors are taken into account by professional computer programmers when selecting the language appropriate for a particular application. Computers will retain the instructions programmed only as long as they are supplied with electricity. It is therefore usual to have a separate means of storing the computer programs so that the power supply can be turned off between use. These program storage devices range from magnetic tapes in cassettes to specialised magnetic ('floppy') disks. The magnetic coating retains the program until it is changed by another magnetic field which occurs when amending the instructions or when writing new ones. The information is stored in the form of binary digits termed 'bits' which are usually arranged in groups of eight or sixteen to form 'bytes'. Floppy disks are readily available in two inexpensive sizes: the smaller (3J/2 in; 8.8 cm) - the so-called 'stiffy', and the larger (5V4 in; 9.9 cm). The latter holds up to 360 000 characters of information (360 kb or kilobytes), the smaller 'stiffy' up to 1 440 000 characters (1.4 Mb or megabytes). In addition, 'hard' disks are available which can store from 20 000 000 to 100 000 000 characters (20-100 Mb). The computer retrieves information from these storage devices by means of an operating system in the form of a special program contained either on the disk or in the computer thus enabling the user to control the procedures. Suitably instructed, the operating system will display information on the visual monitor screen; print it out; capture and store data 'typed' in from the keyboard; and store, retrieve, delete and amend instructions on the associated storage equipment. For bacterial identification by computer, a database containing adequate information about the test characters of a suitable range of known species is first necessary. We describe in Chapter 10 how this information is recorded and transcribed into a form suitable for database compilation together with a simple theoretical example to illustrate the usual methods and statistical procedures used for comparing unknown strains with those in the database. With the large databases necessary for confidence in the results, it is obviously more efficient for speedy bacterial identification to use tailor-made programs prepared with detailed computer instructions. Databases suitable for the identification of medical bacteria have been compiled in many countries

250

and are often used now for tabulating the results in scientific papers. Unlike the diagnostic tables as presented in this Manual with the test results represented by +, -, d and other symbols, database tables express them as percentages or as probabilities for the characters specified (Bascomb et al., 1973; Kelley & Kellog, 1978; Wayne et al., 1980; Feltham & Sneath, 1982; Williams et al., 1983; Feltham, Wood & Sneath, 1984; Bryant et al., 19866). Diagnostic kit manufacturers have used such computer proformas to generate profile registers or lists of medical bacteria characterized in this way. Some have also provided computer programs and the necessary databases for use with their own diagnostic kits. Several computer manufacturers designed their computers for this highly competitive market but their numbers reduced gradually until IBM, the largest computer company, dominated the field with its personal computer in the majority of countries. Its personal computer disk operating system, PC.DOS and, for other computers the equivalent disk operating system MS.DOS or Windows 3 (produced by the company Microsoft), are now used universally as a virtual international standard. The programs called GENMAT (general matrix) containing the material tabulated in this Manual as described by Feltham, Wood & Sneath (1984) are all written in BASIC language for PC or MS. DOS operation. They are therefore suitable for use with most computers and all those compatible with IBM personal computers. To identify bacteria with an MS.DOS BASIC computer identification program such as GENMAT based on the tables in this Manual, the following equipment is suggested: IBM Personal Computer or one compatible with it; 640Kb or greater memory and keyboard; data storage facility with as a minimum dual floppy disks; 80 column dot matrix printer; MS.DOS (3.2 or later version) and BASIC language implementation. Other commercial computer software packages useful in the microbiology laboratory include those for word-processing, spreadsheets, graphics for histograms, pie charts, etc, and databases for literature references, addresses and the like. For clear and simple instructions on how to program a computer in easy stages and in a methodical way with BASIC language, we can recommend 'Computer Programming in BASIC by Carter and Huzman (1987). Computers can also be used in the microbiology laboratory for a broad range of other purposes including the routine data handling requirements of clinical laboratories (Rant, Feltham & Shepherd, 1987). Electronic 'daybooks' together with entry of results and printed reports provide the core of most systems. Quite apart from the classification and identification of bacteria, many laboratories have extended this basic need to monitoring for hospital infection control and surveillance purposes; to the epidemiology

USE OF COMPUTERS and trend analysis of communicable diseases and outbreaks for public health purposes; to the reading and interpretation of manual laboratory procedures such as antibiotic sensitivity testing; and to wide-ranging 'ad hoc' searches on the database for a variety of clinical laboratory purposes. Further, with the increasing use of automated analytical procedures for routine microbiological purposes, such equipment can be connected to or interfaced with computer systems, thus saving transcription time as well as errors. The amount and scope of information stored depends on the particular computer system, but the introduction of recent software environments (the so called '4th generation languages') together with 'total' hospital and healthcare information systems with relational links between departmental databases provides ready and instant access to information on potential nosocomial as well as other infections. The computer communications information ('informatics') explosion has also led to the wide availability of 'viewdata'

in homes, offices and in general practice. Validated data for example on infectious agents and antibiotic resistance can be transmitted electronically from the microbiology laboratory to all out-stations with peripheral display terminals for immediate access by medical practitioners. It should be noted, however, that when personal (including clinical) information is held on file in computer databases (but not in manual data storage systems) then in the UK registration under the Data Protection Act (1984) may be necessary. Advice may be obtained from the Data Protection Registrar, Springfield House, Water Lane, Wilmslow, Cheshire SK9 5AX, UK. Despite this glimpse of the many applications of computers in microbiology, we emphasize again that simple and practical laboratory methods for bacterial identification described in this Manual are bench-orientated and do not require any special equipment or a computer.

251

APPENDIX G The Bacteriological Code

For scientific purposes, the naming of living organisms requires rules to ensure standardization and consistency in nomenclature and thus in the unequivocal communication of identity. The necessary scientific rules for naming all living organisms are embodied in international codes of nomenclature. Separate codes have been formulated for animals, plants, bacteria and viruses. The rules governing the application and use of names for bacteria were long overdue before they were promulgated in the first Bacteriological Code approved in 1947 (Buchanan, St. John-Brooks & Breed, 1948). This code, which was largely based on the Botanical Code, was both long and complicated. Subsequent attempts to increase its usefulness resulted in further difficulties despite the accompanying explanations given in the revised versions published in 1958 (Buchanan et al., 1958) and in 1966. Such rules are necessarily framed in legalistic language, which is not always easily understood. The Bacteriological Code has two basic guidelines: (i) to avoid translation into different languages, all names must be Latinized so as to be clearly recognized both scientifically and internationally and (ii) the names must have definite positions in the relevant taxonomic hierarchy. Informal or vernacular names for bacteria such as 'coliform organisms' and the 'tubercle bacillus' are not governed by the Code although it may help to regulate their use by recommendations for good practice. Informal names such as those for organisms which differ only antigenically are governed by separate rules. Scientific names are used for species, genera and higher ranks. Thus Mycobacterium tuberculosis is the scientific species name for the tubercle bacillus; Staphylococcus is the generic name for the group of related staphylococcal species; and Micrococcaceae the family name for the group of related genera of Gram-positive cocci. Following initial proposals (Lapage et aL, 1973) the most recent revision of the Bacteriological Code - formally entitled the International Code of Nomenclature of Bacteria - was first published by Lapage et al. in 1975. This version, known coloquially as the Revised Code or the

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'1976 revision' authorised a new starting date (1st January 1980 instead of 1st May 1975) for the names of bacteria to be ratified. An initial document, the Approved Lists of Bacterial Names (Skerman, McGowan & Sneath, 1980) listed all names that were nomenclaturally valid from that date. Part of the introduction to this important document is reproduced with permission in Appendix H. Any bacterial names not included in these lists in 1980 lost all nomenclatural standing and therefore official status unless approved and re-instated subsequently in validation lists published as supplements in the International Journal of Systematic Bacteriology (USB). Both the Revised Code and the Approved Lists of Bacterial Names are under the jurisdiction of the International Committee on Systematic Bacteriology, which is assisted by the Judicial Commission in considering alterations to the Code and any exceptions which require amending of specific Rules. All species of bacteria are named in accordance with the principles and rules of nomenclature detailed in the Code. The correct names of species or of higher taxonomic ranks, is determined by three prime criteria: (i) Valid publication All variations to the lists of bacteria, including new names as well as revisions to existing names, are governed by the Code and must be published either primarily in the International Journal of Systematic Bacteriology (IJSB) or, if validly published elsewhere, an announcement of the publication must be made in the IJSB. All descriptions of a named taxon or references to previous effectively published descriptions of it must be given in the IJSB. A formal description of a named taxon must be given and, in the case of cultivable organisms, cultures of the designated type strains of newly named species and subspecies must be deposited in recognized culture collections so that they will be available throughout the world. The essential part of the description of a new organism is a list of characteristic properties that distinguish it from other organisms thus enabling it to be recognized. Minimum criteria for the description of certain groups of

BACTERIOLOGICAL CODE bacteria have been formulated by the Judicial Commission subcommittees; those for other bacterial groups have yet to be completed. These criteria set standards which will ensure not only consistency but also that taxa are not defined unnecessarily thus avoiding repetition of the confusion that existed before 1980. (ii) Legitimacy of the name This forms the main legalistic part of the Code and covers the Latinization of Surnames and of Greek or Latin words which describe or refer to the organisms, e.g. Escherichia coli and Staphylococcus aureus. Taxonomic hierarchy. Each taxonomic level above species is inclusive and the names within it denote successive categories, each of which has a particular position in the hierarchy referred to as its rank. In decreasing order, the main categories of rank are: Kingdom, Phylum, Class, Order, Family, Genus and Species. Form of Names. The form of the latinized names differs with the rank. The species name consists of two parts, the first denoting the genus name and the second, the specific name. Genus names are latinized substantive nouns which are spelled with an initial capital letter, e.g. Shigella. The specific epithet is a latinized adjective spelled with an initial lower case letter, e.g. dysenteriae. Thus the binomial name, or binomen, Shigella dysenteriae means the 'Shigella of dysentery', so named after the Japanese bacteriologist, Shiga. Above the rank of genus, most names are plural adjectives in the feminine gender, e.g. Pseudomonadaceae (Family), Pseudomonadales (Order). Nomenclatural types. These are cultures of bacterial strains which have been deposited in recognized culture collections together with a description of the strain. When a new species is first described the actual strain deposited may not prove subsequently to be typical of the whole group once more members have been identified and characterized. A 'type strain' therefore represents a reference point for the name. There are various categories of 'type strains'. For example, a neotype is a new strain deposited in a culture collection because the original type strain has been lost or is not available. However, not all descriptions of bacteria have a corresponding associated cultural type strain stored for posterity. This applies to many of the medical bacteria that were isolated and categorized in the 19th and early 20th centuries; the original type strain of

Staphylococcus aureus, for example, is no longer in existence. (iii) Priority of publication According to the Rules of the Revised Code, the correct name for a taxon must be the first name validly published in a description of that taxon. This rule still causes some argument among taxonomists as the modern characterization methods available sometimes lead to 'new' species being described though subsequently previous published descriptions are found which had been removed from the Approved Lists either because of poor or incomplete descriptions or because type cultures to match the 'new' organism were not available. Such disagrements are settled by the Judicial Commission with decisions made to reduce confusion. Although the Bacteriological Code, the Approved Lists of Bacterial Names and the Judicial Commission have together done much to settle the majority of scientific nomenclatural problems, a few anomalies persist. Generic or specific synonymns for example, can be perpetuated by scientific preference and continued use or by medical considerations; thus Legionella micdadei is sometimes referred to as 'Tatlockia', and Morganella morganii is still often referred to as Proteus morganii. The Code makes provision for these exceptions and the names retained by international agreement are termed 'conserved'. A 'conserved name' is one which must be used instead of all earlier synonyms. For example, Edwardsiella anguillimortifera is an earlier synonym for the commonly used name Edwardsiella tarda and, according to the rules, the former should be used. However, in view of the common usage of the latter, a proposal has been made to the Judicial Commission to conserve the name E. tarda over E. anguillimortifera. In the case of Morganella morganii and Proteus morganii, both names are in the Approved Lists and are therefore correct but only actual usage will determine which is to be preferred. Knowledge of recent additions to or changes in bacterial nomenclature are important. To help disseminate such information, summaries of the validated changes in nomenclature and additions to the Approved Lists are published in the USB each year. In this way, archaic and non-standard forms of bacterial names are not perpetuated and false nomenclatural trails thus eliminated.

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APPENDIX H The Approved Lists of Bacterial Names

Following the 1976 revision of the Bacteriological Code (see Appendix G), the Approved Lists of Bacterial Names was published in the International Journal of Systematic Bacteriology (USB) on behalf of the Ad Hoc Committee of the Judicial Commission of the International Committee on Systematic Bacteriology (ICSB) of the International Association [now Union] of Microbiological Societies

(Skerman et aL, 1980). It took effect on 1st January 1980 and was subsequently reprinted in book form in 1980 by the American Society for Microbiology. For information, and with permission, the explanatory Introduction to the Approved Lists is reproduced here. All subsequent valid alterations and additions as well as proposals for change are published in the USB.

Approved Lists of Bacterial Names edited by V.B.D. SKERMAN,i VICKI McGOWAN,* AND P.H.A. SNEATH 2 Department of Microbiology, University of Queensland, St Lucia, Queensland 4067, Australia1 and MRC Microbial Systematics Unit, University of Leicester, Leicester LEI 7RH, England 2 on behalf of The Ad Hoc Committee of the Judicial Commission of the ICSB (International Journal of Systematic Bacteriology, vol. 30, pp. 225^20, 1980) INTRODUCTION At the meeting of the Judicial Commission of the ICSB held in Jerusalem on the 29th March, 1973 an Ad Hoc Committee was appointed (Minute 22) to organize a review of the curently valid names of bacteria with the object of retaining only names for those taxa which were adequately described and, if cultivable, for which there was a Type, Neotype or Reference strain available; to compile these names under the title of Approved Lists of Bacterial Names, and to publish the lists in the International Journal of Systematic Bacteriology, to become effective on January 1, 1980. This date would then replace May 1, 1975 (International Code of Nomenclature of Bacteria and Viruses, Rule 10) as the new date for determining priorities for names of new taxa. The members of the Ad Hoc Committee originally appointed to oversee the task were S.P. Lapage, H.P.R. Seeliger and V.B.D. Skerman (Chairman). Following his

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election as President-Elect of IAMS, H.P.R. Seeliger resigned from the Committee and was replaced by J.G. Holt. P.H.A. Sneath, as Chairman of the Judicial Commission, was coopted to the Committee. This Committee was responsible for the editing of the first draft of the Approved Lists of Bacterial Names published in the USB (26, 1976, 563-599). The Ad Hoc Committee was reconstituted by the Judicial Commission during its meetings at the International Congress of the IAMS in Munich in September, 1978, with V.B.D. Skerman (Chairman), P.H.A. Sneath (newly-elected Chairman of the ICSB) and L.G. Wayne (newly elected Chairman of the Judicial Commission) as Committee members. This Committee was assigned wide powers to complete the work on the Lists and arrange publication. The initial Committee agreed that, as a basis for inquiry, all names which had been included in the eighth edition of Bergey 's Manual of Determinative Bacteriology, whether

APPROVED BACTERIAL NAMES listed as recognised species, synonyms, species incertae sedis, or under other headings, should be circulated; that all Subcommittees on Taxonomy of the ICSB should be asked for advice on the retention of taxa for which the Subcommittees were responsible and that specialists be approached for advice on other taxa - only a small list of taxa remained to be considered by the Ad Hoc Committee itself. Advice was also sought on additional names of taxa which had been validly published since the publication of the Manual. Special provision had been made in the Code of Nomenclature for the inclusion of names validly published after the 1st January, 1978 (Rule 24a). A list of specialists was drawn up and approved by the Ad Hoc Committee. Each specialist was asked to associate two others in reaching a decision on name retention. The Chairman accepted the responsibility for coordination of work associated with the selection of generic names and specific and subspecific epithets and P.H.A. Sneath for the names of higher taxa. Names of Genera, Species and Subspecies: The Lists of names of species and subspecies were circulated by airmail in April, 1976 and a draft list of names recommended for retention published in the IJSB (26, 1976, 563-599) together with the list of those people associated with that stage of the project. Reference may be made to the draft list for details of the procedures adopted in processing the information. The object of publishing the draft list was to enable microbiologists, in general, to submit opinions to the Ad Hoc Committee. Several were received. Some of these resulted in modification to lists submitted by Subcommittees on Taxonomy and others. Rule 24a of the Code of Nomenclature provided, inter alia, for the publication with each bacterial name the name(s) of the author(s) who originally proposed it, a reference to an effectively published description of each species and its nomenclatural type whenever possible, and a Type, Neotype or Reference strain by its designation. To conserve space in publication, it was agreed that for all references to the 8th edition of Bergey's Manual (Buchanan, R.E. and N.E. Gibbons (eds). 1974. Bergey's Manual of Determinative Bacteriology, 8th ed. The Williams and Wilkins Co., Baltimore) be contracted to 'Bergey 8'. Although these requirements were formally published in the Code on 1st January, 1976 little action was taken by those responsible for various taxa and in order to assist resolution of the problem, the Chairman and his staff produced a document which listed Collections of Cultures of Microorganisms which held Type, Neotype and Reference

strains for those species whose names appeared in the draft list and subsequent amendments. This list, which will be published elsewhere as a publication of the World Data Center for Microorganisms, has been used as a guide to the deletion of numerous names for which no cultures could be located. The requirements of Rule 24a together with the list of strains, were discussed at a meeting of the ICSB with Subcommittees on Taxonomy at the International Congress for Microbiology in Munich in September, 1978 where the decision was made to list only one designation for each strain and preferably that of the American Type Culture Collection, where available. Because of difficulties associated with locating references to publications in which Type strains, Neotype strains or Reference strains had been designated, it was agreed that references would be given only to those publications concerned with the naming of the taxa and to descriptions of the taxa. The Judicial Commission also determined that publication should be in the January (1980) issue of the IJSB, thus negating the requirement of Rule 24a that publication take place prior to 1st January 1980. This action enabled the inclusion in the Approved Lists of names of all new taxa which had been validated by publication in the October, 1979 issue of the IJSB and so has obviated the need to make any further additions to the Approved Lists of Bacterial Names. Formal notice of these requirements was circulated, by airmail, to all Subcommittees and specialists on 5th February, 1979, requesting return of the information by 31st August, 1979. Meanwhile, the Chairman and his staff proceeded as far as possible with the compilation of the whole of the required information, subsequently making use of such submissions as were ultimately received from others, to verify compilations already made. Computer printouts, in the final format of assembled information, were distributed by airmail to all participants in July, 1979 for the purpose of verification and for the provision of information which was still missing. A final decision to retain or delete some names was based on our ability to obtain the relevant information. Names of Taxa Above the Rank of Genus: Compilation of names of higher taxa was undertaken by Sneath and his associates. No draft list was published but a proposal was discussed at some length in an open meeting at the Munich Congress. The format of the list as submitted was modified to conform with the format adopted for the names of the genera and species. References given in this section of the lists are restricted to those relating to the higher taxa. Those to genera will be found in the list of

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APPENDIX H genera and species. Designation of Strains:

LSU

Strains have been designated by accession numbers of Culture Collections in which they have been deposited. Every effort has been made to ensure that the strains are actually available but no guarantee of this can be given. Names, which have been omitted, may be revived if the location of suitable strains can be ascertained. The following is a list of addresses of institutions, together with acronyms used to assign accession numbers. AMRC FAO-WHO International Reference Centre for Animal Mycoplasmas, Institute for Medical Microbiology, University of Aarhus, Aarhus, Denmark. ATCC American Type Culture Collection, Rockville, Maryland 20852, USA. BKM All-Union Collection of Microorganisms, Institute of Microbiology, USSR Academy of Sciences, Moscow, USSR. BKMW Culture Collection, Institute of Microbiology, USSR Academy of Sciences, Moscow, USSR. CBS Centraalbureau voor Schimmelcultures, Baarn, The Netherlands. CCM Czechoslovak Collection of Microorganisms, J.E. Purkyne University, Brno, Czechoslovakia. CDC Center for Disease Control, Atlanta, Georgia, USA. CIP Collection of the Institute Pasteur, Paris, France. DSM Deutsche Sammlung von Mikroorganismen, Gottingen, Federal Republic of Germany. IAM Institute of Applied Microbiology, University of Tokyo, Tokyo, Japan. IFO Institute for Fermentation, Osaka, Japan. IMRU Institute of Microbiology, Rutgers - The State University, New Brunswick, New Jersey, USA. IMV Institute of Microbiology and Virology, Academy of Sciences of the Ukrainian SSR. INA Institute for New Antibiotics, Moscow, USSR. INMI Institute for Microbiology, USSR Academy of Sciences, Moscow, USSR. IPV Istituto di Patologia Vegetale, Milan, Italy. KCC Kaken Chemical Company Ltd., Tokyo, Japan. LIA Museum of Cultures, Leningrad Research

LMD

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NCDO

NCIB

NCMB

NCPPB

NCTC

NIAID NIHJ NRC NRL

NRRL

PDDCC

TC

UMH UQM

VKM VPI WINDSOR

Institute of Antibiotics, 23 Ogorodnikov Prospect, Leningrad L-20, USSR. Louisiana State University, Baton Rouge, Louisiana, USA. Laboratorium voor Microbiologie der Landbouwhogeschool, Wageningen, The Netherlands. National Collection of Dairy Organisms, National Institute for Research in Dairying, University of Reading, England, UK. National Collection of Industrial Bacteria, Torry Research Station, Aberdeen, Scotland, UK. National Collection of Marine Bacteria, Torry Research Station, Aberdeen, Scotland, UK. National Collection of Plant Pathogenic Bacteria, Plant Pathology Laboratory, Harpenden, England, UK. National Collection of Type Cultures, Central Public Health Laboratory, Colindale, London, England, UK. National Institute of Allergy and Infectious Diseases, Hamilton, Montana, USA. National Institute of Health, Tokyo, Japan. National Research Council, Ottawa, Canada. Neisseria Reference Laboratory, US Public Health Service Hospital, Seattle, Washington, USA. Northern Utilization Research and Development Division, US Department of Agriculture, Peoria, Illinois, USA. Culture Collection of Plant Diseases Division, New Zealand Department of Scientific and Industrial Research, Auckland, New Zealand. Thaxter Collection, Farlow Herbarium, Harvard University, Boston, Massachusetts, USA. University of Missouri Herbarium, Missouri, USA. Culture Collection, Department of Microbiology, University of Queensland, Brisbane, Australia. Institute of Microbiology, Academy of Sciences of the USSR, Moscow, USSR. Virginia Polytechnic Institute and State University, Blacksburg, Virginia, USA. Culture Collection, University of Windsor, Windsor, Ontario, Canada.

APPROVED BACTERIAL NAMES Acknowledgements: Whilst the task of compilation and preparation of manuscripts for publication has been of necessity concentrated in two areas, the successful completion of the task would not have been possible without the associated efforts of a large number of people whose names are recorded here. T. V. Aristovskaya, P. K. C. Austwick, E. Baldacci, L. Barksdale, E. Barnes, P. Baumann, J. H. Becking, E. L. Biberstein, I. Bousfield, K. Bovre, W. J. Brinley-Morgan, D. Brenner, T. Brock, E. Brockman, M. Bryant, J. C. Burnham, J. Carr, W. Catlin, E. P. Cato, D. Claus, G. Colman, R. R. Colwell, M. J. Corbel, K. T. Crabtree, T. Cross, G. A. Dubinina, P. Dugan, D. W. Dye, K. Eimhjellen, G. Eldering, W. H. Ewing, S. Faine, S. M. Finegold, E. A. Freundt, W. Frederiksen, A. L. Furniss, E. Garvie, N. E. Gibbons, M. Goodfellow, M. Gordon, B. V. Gronov, H. Hatt, A. C. Hayward, M. Hendrie, S. D. Henriksen, A. Henssen, G. J. Hermann, G. Gobbs, L. V. Holdeman, R. Hugh, D. B. Johnstone, D. Jones, L. V. Kalakoutskii, O. Kandler, K.-A. Karlsson, R. M. Keddie, M. Killian, K. Kitahara, J. Knapp, W. Knapp, M. Kocur, N. R. Kreig, S. P. Lapage, J. M. Larkin, H. Lautrop, H. A. Lechevalier, J. DeLey, R. Mocci, S. Maier, N. S. Mair, P. H. Makela, M. Mandel, E. McCoy, H. D. McCurdy; K. McNiel, W. E. C. Moore, N. Nishida, H. Nonomura, R. A. Ormsbee, F. Orskov, I. Orskov, E. van Oye, O. M. Parinkina, N. Pfennig, J. E. Phillips, F. Pichinoty, M. Pittman, T. Pridham, C. I. Randies, J. W. M. LaRiviere, M. Rhodes-Roberts, D. S. Roberts, R. Rohde, B. Rowe, E. B. Rozlycky, E. Runyon, R. Sakazaki, V. Scardovi, D. Schafer, R. J. Sidleer, J. M. Shewan, G. C. Simmons, I. J. Slotnick, R. M. Sibert, P. H. Smith, J. T. Staley, M. Starr, Y. Terasaki, Y. T. Tchan, E. Thai, J. Thompson, H. Truper, G. Tunevall, D. C. Turk, M. Veron, N. Walker, S. W. Watson, G. Wauters, L. Wayne, R. E. Weaver, O. B. Weeks, E. Weiss, H. J. Welshimer, S. Williams, G. A. Zavarzin, J. G. Zeikus, K. Zinneman. Thanks are also due to Miss Annette McLennan who typed the original manuscripts and to Dr Lindsay Sly, Dr Horst Doelle and Mrs Elizabeth Marden for considerable assistance in the search for Culture Collections maintaining strains of taxa in the Approved Lists and other information and to the numerous other unnamed people who have assisted in various ways in laboratories throughout the world. The computer programs used in compiling these lists were written and controlled by Mr. Geoffrey Dengate, Systems Programmer, Prentice Computer Centre, University of Queensland. His help and expertise are sincerely acknowledged.

Financial assistance of the following is gratefully acknowledged: Bergey's Manual Trust, Deutsche Gesellschaft fur Hygiene und Mikrobiologie, International Association of Microbiological Societies, International Union of Biological Sciences, Society for Applied Bacteriology, Society for General Microbiology, and the United Kingdom Federation of Culture Collections. SPECIFIC INFORMATION RELATING TO THE APPROVED LISTS OF BACTERIAL NAMES The names included in these Lists are those selected by Subcommittees on Taxonomy of the ICSB, specialist advisers and, where the former have not been available or have declined to advise, by the members of the Ad Hoc Committee. With few exceptions (vide infra) names which have been included in the Lists are those which: were validly published before the 1st January, 1978 and which have been listed by advisers. have been validly published since the 1st January, 1978 by publication in the USB or by inclusion in the lists of new names cited in the USB (to and including Volume 29, Part 4, 1979) as having been effectively published in other Journals. Specific attention is drawn to the following. 1. Names included in the Approved Lists of Bacterial Names are the only names which are nomenclaturally valid as at the 1st January, 1980. All other names which have appeared in the literature prior to 1st January, 1980 are nomenclaturally invalid. Names which have been effectively published in Journals other than the USB but which have not been cited in the lists of new names published in the USB, have not been validly published under Rule 27 of the Code of Nomenclature. Such names may be validated by publication in forthcoming issues of the USB and will simply constitute names of new taxa. 2. No name appearing in the Approved Lists of Bacterial Names is conserved in the sense defined under Rule 56b of the Code of Nomenclature (1976 Revision). 3. Any name previously rejected by the Judicial Commission remains a nomen rejiciendum and can no longer be used to name a bacterium. 4. By majority decision of the Ad Hoc Committee, supported by an overwhelming majority of the members of the ICSB, all strains of bacteria which were submitted as nomenclatural Type strains, Neotype strains, Proposed

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APPENDIX H Neotype strains or Reference strains have been elevated to the status of Type strains. The few objections which were received to the elevation of reference strains to Type status were based on concern that such reference strains may not prove to be the best choice for Type strains. This objection however, applies equally to quite a number of Type strains, particularly monotypes of which there are, and will continue to be, several. It seemed more appropriate that names appearing in the Approved Lists of Bacterial Names, which constitutes a new base for future nomenclature, should have Type status or be omitted. 5. For some names included in the Lists no strains were available. These names were retained to preserve the status of A. some microorganisms for which habitats are well known, which have distinct morphological characters but which have never been isolated. In most cases only the Type Species has been retained to preserve the status of the genus. B. a genus which contained well described species for which representative strains were available for all but the Type Species of the genus itself. Exclusion of the Type Species would have necessitated the exclusion of the genus as a whole under Rule 20c of the Code of Nomenclature. There were several of these. It is hoped that early action will be taken to request the Judicial Commission to conserve these genera with new Type Species in order that each species within these genera will be represented by a Type strain in a recognised culture collection. 6. The Ad Hoc Committee has repeatedly stressed that its task was nomenclatural and not taxonomic; further that Subcommittees and others were not required to solve taxonomic problems before advising on retention of names. They were requested to omit names of doubtful species as such species could be revived (see 8 below). Because there are some differences of opinion regarding the generic position of some species it has been necessary to include the same microorganism under two names, each with the same specific epithet and the same nomenclatural type strain. In such instances cross-reference has been made to the alternate name. This applied specifically to the species in the genus Beneckea and the genus Vibrio and to a few instances in the Enterobacteriaceae. For this reason it is necessary here to reaffirm the statement made previously that no name in the Approved Lists of Bacterial Names has been conserved. Names in the Lists may later be conserved, rejected, merged with other names or subjected to any other valid nomenclatural act.

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7. The names of some genera, for which there are no available strains for the Type Species, have been omitted from the Lists in order to permit the resolution of a very difficult nomenclatural situation arising from the recent publication of provisional names which have no standing in nomenclature but which have been cited by subsequent authors as though they were valid. Addition of new species, with nomenclatural types, to an invalidly published generic name renders the new names equally invalid. This refers specifically to the methane oxidising bacteria, where the otherwise valid name Methanomonas has been omitted in the hope that by so doing the impasse caused by the illegitimate substitution of the name Methylomonas may be resolved. Specialists in this field are now urged to publish a fully validated list of appropriate species. 8. Reuse and revival of names which do not appear in the Approved Lists of Bacterial Names is authorized by Rule 28a. In addition, the Code of Nomenclature (1976 Revision) contains a number of Provisional Rules. At the time of publication of this List these Rules still have only provisional status. It will require a specific act on the part of the Judicial Commission at a formally constituted meeting to raise these Provisional Rules to Rule status. Meanwhile they continue to be inapplicable. Revival of Names: A name which has not appeared in the Approved Lists of Bacterial Names and which has not been listed as a nomen rejiciendum by the Judicial Commission may be revived {nomen revictum) if it is to be used for the same taxon to which (in the author's opinion) it was originally applied. For citation of such names see Rule 28a and Provisional Rules B2 and B3. As most names which have not been included in the Approved Lists of Bacterial Names have been omitted because of uncertainty of the validity of the taxa to which they apply, the revival of names should be practised very conservatively. Reuse of names - Specific and Subspecific epithets: There never has been any rule which forbids the use of the same specific epithet for taxa within different genera. Many contributors to the Approved Lists of Bacterial Names have voiced considerable concern at the possibility that a specific epithet may be used within the same genus as that in which it was originally used but for the naming of a taxon to which it was not originally applied. Unless the Judicial Commission takes action to prevent such usage, the Ad Hoc Committee can only urge taxonomists to avoid such reusage as may lead to considerable confusion in future nomenclature.

APPROVED BACTERIAL NAMES For the same reason taxonomists are urged not to use as a specific epithet within a genus a name which has been used for an infrasubspecific subdivision of a species within that genus. This applies particularly to several genera of predominantly plant pathogens, where previously cited species have been relegated to the status of pathovars within species listed in the Approved Lists of Bacterial Names. As such infrasubspecific pathovars may later be elevated to the status of subspecies or species, preemption of the pathovar name as a specific or subspecific epithet of a different taxon would obviously cause confusion. The International Society for Plant Pathology is publishing, in the Review of Plant Pathology, a list of pathovars of species which appear in the Approved Lists of Bacterial Names. Taxonomists are urged to consult this list before proposing new specific epithets or reusing or reviving old epithets for new species within the relevant genera. Reuse of generic names: The reuse of a generic name which has not appeared in the Approved Lists of Bacterial Names and which has not been listed as a nomen rejiciendum by the Judicial Commission does not appear to be fraught with the same difficulties as those associated with specific and subspecific epithets. Taxonomists are never-

theless asked to seriously consider the possibility of confusion arising from the use of such names before taking such action. The correct form of citation for reused names is given in Provisional Rules B1 and B4. 9. Omissions from Approved Lists of Bacterial Names. No names will be added to or removed from the Approved Lists of Bacterial Names. Attention may be drawn to errors in nomenclature or to errors in spelling or citations of references and such communications should be directed to the Executive Secretary, ICSB, for transmission to the appropriate body. Please note that the omission of diacritical signs was intentional and dictated by the limitations of the computers used in setting up the type. These omissions do not constitute 'errors' in the above sense. Every effort has been made to obviate errors by reference of the computer printouts of the final information to the original advisers for correction and also for insertion of some missing information. The final printout prior to production of the bromide copy for printing has been checked by the Chairman but the magnitude of the task allowed only of a search for the more glaring errors of which there were fortunately few.

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APPENDIX I Reconciliation of approaches to bacterial systematics

In Section 8.4 we discussed briefly the impact of numerical taxonomy and the newer genetic techniques for measuring bacterial relatedness and evolutionary divergence on traditional classification and nomenclature as used in this Manual. Because of their fundamental significance for taxonomy in the future we reproduce in full, with permission from the International Committee on Systematic Bacteriology, the report of the ad hoc committee set up to consider the various approaches to bacterial systematics and their relationship to each other published in the International Journal of Systematic Bacteriology by Wayne et al. (1987) on behalf of the International Union [formerly Association] of Microbiological Societies.

Report of the Ad Hoc Committee on Reconciliation of Approaches to Bacterial Systematics L. G. WAYNE,!* D. J. BRENNER,2 R. R. COLWELL,3 P. A. D. GRIMONT,4 O. KANDLER,* M. I. KRICHEVSKY,* L. H. MOORE,? W. E. C. MOORE,? R. G. E. MURRAY,8 E. STACKEBRANDT,* M. P. STARR,io AND H. G. TRUPERU. Veterans Administration Medical Center, Long Beach, California1; Centers for Disease Control, Atlanta, Georgia2; University of Maryland, College Park, Maryland3; Institut Pasteur, Paris, France4; University of Munich, Munich, Federal Republic of Germany5; National Institute of Dental Research, Bethesda, Maryland6; Virginia Polytechnic Institute and State University, Blacksburg, Virginia7; University of Western Ontario, London, Ontario, Canada8; University of Kiel, Federal Republic of Germany9; University of California, Davis, California10; University of Bonn, Bonn, Federal Republic of Germany11

Bacterial taxonomy, which began as a largely intuitive process, has become increasingly objective with the advent of numerical taxonomy and techniques for the measurement of evolutionary divergence in the structure of semantides, i.e., large, information-bearing molecules such as nucleic acids and proteins. These developments have forced the adoption of changes in nomenclature, sometimes with disruptive or even perilous consequences (2). Since the present nomenclatural system evolved during a period when hierarchial taxonomic divisions were only vaguely defined, it has become important to reexamine that system in the light of newer taxonomic understanding. Accordingly, an ad hoc committee of the International Committee for Systematic Bacteriology was convened for a Workshop on Reconciliation of Approaches to Bacterial Systematics at the Institut Pasteur, Paris, on 14 to 16 May 1987. Perspectives. To arrive at a common ground of understanding, the committee reviewed the current state of bacterial systematics from the following perspectives: (i) Phylogenetic. Phylogenetic studies are directed at a basic understanding of pathways through which taxa have evolved from primordial and recent ancestors, calculated from analyses of evolutionary distances between selected semantides. (ii) Descriptive. Descriptive research represents the bridge between semantide-based phylogenetic taxonomy and traditional phenotype-based bacterial systematics. Organisms are described in phenotypic terms, and the descriptions help define a taxonomic group that may also be recognized at the phylogenetic level. (iii) Diagnostic. Diagnosis involves the selection of features from those in a phenotypic description, or of probes derived from phylogenetic analyses, in a way that permits the recognition of an unknown strain and the assignment of a label to it. The most useful labels for applied purposes are names at the genus, species, and subspecies levels; infra-

* Comments on the conclusions and recommendations included in this report are welcome and should be sent to Lawrence G. Wayne, Tuberculosis Research Laboratory (151), Veterans Administrations Medical Center, 5901 East Seventh St., Long Beach, CA 90822.

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RECONCILIATION OF APPROACHES TO BACTERIAL SYSTEMATICS subspecific categories that are not governed by the Bacteriological Code (1) may be useful for recognition of special attributes. (iv) Associative. Associative studies use a name to evoke practical information about a strain, such as its medical, industrial, or ecologic significance. The committee offers the following conclusions and recommendations. HIERARCHICAL TAXONOMY An ideal taxonomy would involve one system. A single formal overall system appears to be adequate, and a second system is not needed if vernacular grouping is retained for diagnostic purposes. There was general agreement that the complete deoxyribonucleic acid (DNA) sequence would be the reference standard to determine phylogeny and that phylogeny should determine taxonomy. Furthermore, nomenclature should agree with (and reflect) genomic information. The group agreed that hierarchical consistency is essential but recognized that the depth in a ribonucleic acid (RNA) dendrogram at which a given hierarchical line is to be drawn may vary along different major branches of the dendrogram and will be strongly influenced by phenotypic consistency at each level. This is a consequence of differences in ages of the different branches and the recognition that older branches will exhibit greater nucleic acid structural genetic drift, even as environmental constraints tend to limit phenotypic drift. A cautionary note about hierarchical interpretation was expressed with the recommendation that active searches continue for additional powerful semantides independent of the ribosomal RNA cistrons. The chemotaxonomic approach has two sets of derivative data: structural phenetic (including "signatures") and phylogenetic (evolutionary). There should not be further designation of hierarchical levels without substantial chemotaxonomic and sequence data to support the proposal. Species, subspecies, and infrasubspecific categories. At present, the species is the only taxonomic unit that can be defined in phylogenetic terms. In practice, DNA reassociation approaches the sequence standard and represents the best applicable procedure at the present time. The phylogenetic definition of a species generally would include strains with approximately 70% or greater DNA-DNA relatedness and with 5°C or less &Tm. Both values must be considered. Phenotypic characteristics should agree with this definition and would be allowed to override the phylogenetic concept of species only in a few exceptional cases. It is recommended that a distinct genospecies that cannot be differentiated from another genospecies on the basis of

any known phenotypic property not be named until they can be differentiated by some phenotypic property. Subspecies designations can be used for genetically close organisms that diverge in phenotype. There is some evidence, based on frequency distribution of A7 m values in DNA hybridization, that the subspecies concept is phylogenetically valid and can be distinguished from the infra-subspecific variety concept, which is based solely on selected "utility" attributes, but not demonstrable by DNA reassociation. There is a need for further guidelines for designation of subspecies. Genera. Genera form the essential basis of bacterial systematics, and each genus must remain subject to continuing assessment. A degree of flexibility in circumscription is necessary. Unfortunately, there is currently no satisfactory phylogenetic definition of a genus. The scope of the definition may differ among genera, as for any other higher taxa. Families. Families can be retained as long as they are consistent in terms of chemotaxonomic and hybridization or sequence data. Any other hierarchical level established between family and division must also be consistent in terms of supporting taxonomic data. Divisions. It is clear that there are at least 15 clearly separated lineages of great antiquity in evolutionary terms at the level of division or greater. Kingdom. There is no need at this time for more than one kingdom, but there may be need for a term to describe major primary lineages. A rank such as phylum may be needed in the future. NOMENCLATURE After a review of alternative approaches to the occasional conflict between binomials based on strict phylogenetic relationships at the species level and the practical application of the binomials, the following recommendations were made. Nomenclature should reflect genomic relationships to the greatest extent possible. Rare exceptions are sanctioned under the emendation to Rule 56a embodied in Minute 7i of the Judicial Commission (Int. J. Syst. Bacteriol. 31:85-87, 1987), introducing the concept of a unomen periculosum". It is anticipated that this concept need be applied in only a limited number of cases and hoped that this type of problem will disappear as new taxonomic methods and interpretations are more universally applied. A mechanism is needed for broad dissemination of information about those cases where such nomenclatural discrepancies have been sanctioned. As needed, various vernacular appendices that pertain to needs for special purposes (medical, veterinary, agriculture, industry, etc.) including information about traits coded by plasmids, phages, and other extrachromosomal agents

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APPENDIX I should be applied. These infrasubspecific "utility" categories are not governed by the Bacteriological Code. In certain medical situations, identification of the presence of certain virulence factors may be more important than the species name. Regarding inadequate nomenclature, it is recommended that formal nomenclature is needed for the major divisional groupings. "Gram positives" should be Firmicutes as presently defined. The term "purple bacteria" causes confusion because it includes more than phototrophic bacteria, which is good reason for a formal name to be established. OTHER CONCERNS Special difficulties are expected in accomplishing the taxonomic reorganization of the major phylogenetic taxon listed as "the purple photosynthetic bacteria and their relatives" (3), and this includes most of the classically defined gramnegative genera, both photosynthetic and chemosynthetic, in the alpha, beta, gamma, and delta groups of the purple bacteria. Thus, the major task of the greatest practical importance is the development of a taxonomic and nomenclatural approach to resolving the linked phylogenetic lineages of the remarkably diverse metabolic types of organisms within each of the groupings. Urgency is dictated by the large number of bacteria involved and by the many species that are crucial in ecological and diagnostic bacteriology. A number of genera must be reassessed, although many will persist in part, if not as a whole. There are gaps in our knowledge because the molecular/genetic surveys are incomplete. The first task is the definition and phylogenetic circumscription of the genera in this phylogenetic taxon, with inclusion of type strains of species in the genera. It is recommended that this problem be assigned for special study by an ad hoc subcommittee of the International Committee on Systematic Bacteriology. Research should be directed toward solving the growth and diagnostic problems of noncultivable or hardly cultivable organisms, e.g., cholera somnicells, nongerminable endospores, and organisms not cultivable axenically but cultivable in multiorganism systems, such as representatives of consortia, Pasteuria, Planctomyces, etc. The Code may have to be emended to clarify the status of multiorganism cultures as type material; e.g., see Rule 18a and Advisory Note C, Chapter 4(1).

A whole base line of bacterial taxonomy of various ecosystems is urgently needed, as is a broader knowledge of species distribution in ecosystems and improved knowledge of little known bacteria. ("Less than 20% of the bacteria are known.") Encouragement must be given to systematic studies and the search for new chemotaxonomic markers. Recognition of the importance of effective taxonomic understanding to all fields, basic and applied, and especially to biotechnology has been slow to develop, although the need is great. The group recognized, also, that ecologically relevant characterization of the members of complex bacterial populations requires the identification of these characters in a burgeoning field of biochemical/ molecular/genetic research. An overall concern of members of the Committee was that any phylogenetically based taxonomic schemes that result must also show phenotypic consistency. ACKNOWLEDGMENTS The International Committee on Systematic Bacteriology and the members of the Ad Hoc committee are grateful to the Bacteriology Division of the International Union of Microbiological Societies, to the International Council of Scientific Unions, and to the Bergey's Manual Board of Trustees for the provision of funds supporting the meeting, and to the Institut Pasteur (Paris) for its hospitality. LITERATURE CITED 1. Lapage, S. P., P. H. A. Sneath, E. F. Lessel, V. B. D. Skerman, H. P. R. Seeliger, and W. A. Clark (ed.). 1975. International code of nomenclature of bacteria. 1976 Revision. American Society for Microbiology, Washington, D.C. 2. Williams, J. E. 1983. Warnings on a potential for laboratory-acquired infections as a result of the new nomenclature for the plague bacillus. Bull. WHO 61:545-546. 3. Woese, C. R., E. Stackebrandt, T. J. Macke, and G. E. Fox. 1985. A phylogenetic definition of the major eubacterial taxa. System. Appl. Microbiol. 6:143-151.

International Journal of Systematic Bacteriology, Oct. 1987, p. 463—464 0020-7713/87/040463-02$02.00/0 Copyright © 1987. International Union of Microbiological Societies

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Vol. 37. No. 4

APPENDIX J Glossary Jl Taxonomic terms J2 Abbreviations and Acronyms

Jl TAXONOMIC TERMS We have been unable to avoid the use of some taxonomic terms in this Manual, and short notes on some of those more commonly used are given below. Those who would like more help or greater detail should consult A Dictionary ofMicrobial Taxonomy by Cowan (1978). Accession number. The number allotted to a culture when it is accessioned (accepted) into a permanently established culture collection. Even if the classification (and the name) of the organism changes, the accession number remains the same. Antibiogram. A record of the sensitivity or resistance of an organism to the different antibiotics listed. It is often an essential part of the bacteriological report made to the clinician who sent the specimen. Sometimes the sensitivity or resistance to particular antibiotics can aid in identification although the possibility of changes during antibiotic therapy should always be remembered. Non-therapeutic substances such as lysozyme or O/129 may also assist in the identification of an organism. Carboxyphilic; capnophilic. Used to describe an organism whose growth is improved, or made possible, by an increase in the CO2 content of the atmosphere. Category. Used in taxonomy to indicate RANK in a hierarchical system of classification: genus, species, and so on. Often also used with the ordinary meaning of 'kind'. Chemotaxonomy. A term used to describe the chemical nature of the structure and functions of organisms as applied to their taxonomy. Conserved Name is one which the Judicial Comission, with reasons, has ruled must be used instead of all earlier synonyms for that taxon. D r Value. A term used in numerical taxonomy to express

the total difference between two strains, taking into account the growth rates and the temperature and duration of incubation. Dendrogram. A tree-like figure used to represent a hierarchy (and lines of descent) of organisms or a diagram showing the different levels of similarity among organisms. Description. The characters of a taxon. When a description has been well written and includes all the essential information, it should enable future workers to recognize the taxon, should they meet it in practice. Descriptions vary greatly in value; older ones, because of the limited knowledge at the time they were written, are often inadequate by present standards, and the taxa may not now be recognizable. Diagnosis in systematic bacteriology (and taxonomy) is a brief statement of characters that distinguish an organism from its neighbours. It is the equivalent of the term 'differential diagnosis' in clinical medicine. Diagnostician. In a medical bacteriology laboratory this means the person(s) responsible for identifying an organism. It does not mean the person(s) responsible for making a clinical diagnosis or the clinician who sent the specimen to the laboratory. Effective publication is the term used in the Bacteriological Code for the kind of publication authorized by it. Effective publication consists of publishing relevant information in printed form for distribution to the public and scientific institutions by sale or gift. Reading a paper at a meeting of a learned society does not constitute effective publication for names of bacteria, but publication in the Proceedings (or Transactions) of the Society concerned may well do so. Publication in the USB constitutes effective publication for new species (see valid publication). Family (and subfamily) represent biological ranks between those of Order and Genus.

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APPENDIX J Genus (genera). One of the basic ranks in hierarchical systems of biological classification between families and species. A genus represents a collection of species with many characters in common though the actual limits are a matter of individual judgement.

formed or published in accordance with the rules of the Bacteriological Code.

G+C ratio. The proportions of the four DNA bases guanine (G), cytosine (C), adenine (A) and thymine (T). Because of the molecular equivalence of G+C to A+T, the ratio is usually expressed as the percentage of G+C to the total of bases (% G+C; moles % G+C).

Infrasubspedfic. A name given to a taxon below the rank of subspecies but which is not subject to the rules of the Bacteriological Code.

Genotype, -ic. The hereditary potential of an organism. A theoretical unit from which existing organisms have developed under the influence of their surroundings and possibly changed character to become the PHENOTYPE. Group. A common term for a collection of similar objects or biological entities. It is used in taxonomy for a collection of strains not yet allocated to a particular RANK; it may be a collection of strains (eventually they may form a species) or a collection of species (perhaps a potential genus). In serology it may be a collection of serotypes. It preferably is written or printed (in English) with the lower case letter g so as not to be confused with defined Groups. Halophil(e) describes an organism that can tolerate and grow in a medium with a salt concentration greater than 0.85%; strictly it should be applied only to organisms with a requirement for a concentration of salt greater than 3%. Homology. A term used to describe the extent of hybridizing capability between the DNA of microorganisms as a percentage. Hybridization describes the extent of reassociation of single-stranded DNA derived from different strains to form double stranded DNA. A high degree of hybridization indicates genetic relatedness. Hyperspace. A term used in numerical taxonomy to represent the dimensions needed to plot the distances between characters in an imaginary space. The closer the points are together, the more characters the organisms share in common. Hypersphere. A term used in numerical taxonomy to represent a cluster of points (OTUs) defined in a hyperspace and distributed in the form of an imaginary theoretical sphere. Illegitimate name of an organism is one that has not been 264

Incertae sedis. A term used for named organisms of uncertain taxonomic position and applied to species and genera.

Legitimate name applied to an organism is one that has been formed and published in accordance with the rules of the Bacteriological Code. Mesophil(e). A term used for organisms that are able to grow over a wide temperature range though not below 8 °C or above 45 °C. Many psychrophilic organisms can grow at temperatures within this range, but mesophiles are not tolerant of cold. Neotype. The author of a new name (of a bacterium) should designate a nomenclatural type strain (see TYPE). If this is not done, a subsequent author may designate a type strain; a neotype is such a designated strain provided it is not one of the strains used by the original author of the name. There are official recommendations for the choice, proposal, and designation of type strains; the old Recommendations in rule 12e of the 1958 edition of the Bacteriological Code were unintentionally omitted from the 1966 version printed in the International Journal of Systematic Bacteriology (USB), 16, 459-90. They are now dealt with fully in the revised Bacteriological Code (1976 revision) in Rule 18e. The most important points for an author who intends to propose a neotype are (i) the proposal must be published in the IJSB; (ii) it must be accompanied by a description (or a reference to a published description) of the proposed strain; and (iii) it must include a record of the deposition of the strain in a permanently established culture collection so that it is readily available. These requirements, however, together with the Approved Lists of Bacterial Names and with the methods currently available for the preservation of bacteria make it unlikely that there will be much need for neotype strains in future. Nomen periculosum. The term for a name validly proposed and taxonomically correct for a species or taxon but which, if accepted, could cause uncertainty, confusion or danger in practice. For example, the proposed name Yersinia tuberculosis subsp. pestis for the plague organism was overruled because it could be medically misleading; the correct name accordingly remains Yersinia pestis.

GLOSSARY Operational Taxonomic Unit (OTU). A term used in relation to work in progress embracing an individual organism, a population of organisms, a taxon, a species or even a genus. In microbiology it is used by numerical taxonomists as a useful substitute for strain(s).

genera of that family and the species of those genera. And as in an army, the species are subordinate to taxa of higher rank. An alternative analogy is to the shelves of a bookstack; families near the top, genera in the middle, and species lower down.

Phase should be reserved for phenomena with cyclical or to-and-fro variation, as for example the phases of Salmonella H antigens.

Similarity coefficient (S value) is a measure, usually expressed as a percentage, of the overall similarity of two strains.

Phenon. A term used for a group of organisms in which the extent of their similarity is established by taxonomic methods. Instead of referring to species and genera, the bacterial groups are defined as phenons with a particular percentage of similarity (e.g. the 85% phenon).

Subspecies. A category of rank immediately below that of species. It is the lowest rank to which the rules of the Bacteriological Code apply.

Phenotype, -ic. The characters of an organism that can be determined now; cf. the GENOTYPE, which is the theoretical starting point that contains the hereditary potential of the taxon. Phylogeny. A term used to describe the expression of evolutionary relationships between organisms.

Sphere (see also Hypersphere). A term used in numerical taxonomy to represent the concept of a spherical cluster of organisms with an operational taxonomic unit (OTU) close to or at the geometrical centre of the cluster. Symbiosis. Often used to mean two organisms living together for mutual benefit. Strictly, symbiosis means physical contact between organisms, each of which is a symbiont. Examples of symbiosis are Rickettsia prowazekii and Pediculus corporis. See also SYNERGISM.

Plasmid. A particle of genetic material (DNA) which contains recognizable genes which can be transferred to another organism. Antibiotic resistance, for example, may be 'plasmid-mediated' thus signifying that the gene for such resistance can be transferred from one bacterium to another. A wide range of biochemical characteristics may be plasmid-borne.

Synergism. The state in which two or more organisms form an organization, sometimes called an ecosystem, for mutual benefit (often called SYMBIOSIS, q.v.). The satellitism shown by colonies of Haemophilus influenzae near colonies of Staphylococcus aureus growing on blood agar is a one-sided form of synergism.

Probes for DNA or RNA are pieces of nucleic acids, derived from known organisms and labelled in some identifiable way, which are used to seek and bind to stretches of DNA or RNA that have complementary molecular base sequences. The proportion of labelled probe thus hybridizing gives a measure of the extent of genetic relatedness, which itself can be used as a taxonomic tool.

Synonym. A word with the same meaning as another; synonyms are different words for the same thing. In nomenclature, synonyms are different names applied to the same taxon, but this does not mean that the individual units or organisms making up the taxon are identical. Strictly, synonymy applies only to the names; taxa and individual bacteria cannot be synonymous.

Psychrophil(e) describes an organism that grows at temperatures below those used in most medical laboratories; the optimal temperature for psychrophils is usually about 20 °C. Organisms able to grow below 5 °C may be described as psychrotrophic.

Systematics. A term used to embrace the many different approaches to the characterization, differentiation, labelling and orderly arrangement of organisms so that the information can be communicated succinctly.

Rank. In a hierarchical system of classification organisms are arranged in a series of ranks (or CATEGORIES) each of which includes all members of the ranks immediately below. A taxon with the rank of family includes the lower taxa with the rank of genus, and each genus is made up of taxa with the rank of species; thus a family consists of the

T m Value. The temperature at which a dilute solution of double stranded nucleic acid melts as indicated by increased light absorbance at a wavelength of 260 nm. Taxon (pi. Taxa). A taxonomic group of any size greater than a single strain. Two or more similar strains can form a taxon; its RANK may be that of a serotype, species, genus,

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APPENDIX J or even a kingdom, with numerous intermediate ranks between kingdom and genus, none of which may have practical significance. Thermophil(e). A descriptive term applied to organisms capable of growing at temperatures above that of animals, usually not below 40 °C. Type. This common English word meaning 'kind' has a special use in taxonomy for the nomenclatural type of a taxon. The nomenclatural type of a species is a strain (the type strain); of a genus it is a species (type species), and of a family or higher taxon the type is a genus (type genus). The nomenclatural type is a means of attaching a name to a taxon; strictly it refers to a particular strain for which that name is published, but other strains that are regarded as similar (and that may be a matter of opinion) are entitled to bear the same name. It is a purely nomenclatural device and it is naive to think that the word type should be used only in its strict taxonomic sense. On the other hand it is reasonable to expect that, in papers on taxonomic subjects, the word will be used only in that strict sense. Valid publication is publication (of a bacterial name) in accordance with certain requirements laid down in the Bacteriological Code. Variant. A strain that shows one or more differences in characters from the parent; largely replaced by the word mutant when the change is believed to be due to a genetic change. Variety ( var.) is a term used for a variant that has become stabilized as a regular form. A category of rank below that of species. It equated previously with subspecies but it cannot be applied to new bacterial names published after 1976. It can, however, be used to indicate an infrasubspecific taxon and is then subject to the rules of the Bacteriological Code.

ALM

ANSI API ASB ASS ATCC

BASIC BCG BCYE BIP BSS CAMP CDC CDSC CLED COBOL CSU CYLG DCA DMRQC DOS EDTA EHC FEMS FN HTST IAMS

J2 ABBREVIATIONS AND ACRONYMS An expansion of some less well-known abbreviations and acronyms may be useful to readers of this Manual. The list is based partly on queries raised by the proof-readers for this edition and is not exhaustive.

IATS ICSB USB IMViC

ACES

buffer - N-2-acetamido-2-aminoethanesulphonic acid albumin + glucose (dextrose) + catalase Acid fast bacilli

ADC AFB

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IUMS KIA

Anaerobe Laboratory Manual, Virginia Polytechnic Institute, Blacksburg, Virginia, USA American National Standards Institute Identification kit system as originally described by Buissiere & Nardon (1968) Aerobic spore-bearing organisms (Bacillus) Ammonium salt sugars media American Type Culture Collection, 12301 Parklawn Drive, Rockville, Maryland 20852, USA Beginners' All-purpose Symbolic Instruction Code (computer language) Bacillus Calmette-Guerin Buffered charcoal yeast-extract medium Bacterial Identification Profile Buffered single substrate media Test described by Christie, Atkins & MunchPetersen(1944) Centers for Disease Control, Atlanta, Georgia, USA Communicable Disease Surveillance Centre, PHLS Cystine-lactose-electrolyte deficient medium Common Business Oriented Language (computer language) Carbon source utilization tests Casein yeast lactate glucose medium De(s)oxycholate Citrate agar medium Division of Microbiological Reagents and Quality Control, PHLS Disk operating system Ethylenediaminetetra-acetic acid (also known as Sequestric acid) Ethylhydrocuprein (optochin) Federation of European Microbiological Societies Fluorescence-denitrification medium of Pickett & Pedersen (1968) High temperature, short time heat-treatment International Association of Microbiological Societies International Antigenic Typing Scheme International Committee for Systematic Bacteriology International Journal of Systematic Bacteriology Indole, methyl red, Voges-Proskaiier, and citrate utilization tests International Union of Microbiological Societies (formerly IAMS) Kligler iron Agar medium

GLOSSARY LJ LV MR MUMPS

Lowenstein-Jensen medium Lecithovitellin Methyl Red test Massachusetts General Hospital Utility Multi-programming System (computer language) NAD Nicotinamide adenine dinucleotide (V factor) NAG non-agglutinable vibrio NCTC National Collection of Type Cultures, PHLS NCV non-cholera vibrio NLF Non-lactose fermenter OF Oxidation-Fermentation test of Hugh & Leifson (1953) ONPG o-nitrophenyl-p-D-galactopyranoside OTU Operational Taxonomic Unit PHB Poly-6-hydroxybutyrate (used in agar medium) PHBA Poly-6-hydroxybutyric acid (characteristic build-up in Pseudomonas cells) PHLS Public Health Laboratory Service, 61 Colindale Avenue, London NW9 5DF, UK PPLO Pleuropneumonia-like organisms PRAS Pre-reduced anaerobically sterilized media PV Panton-Valentine leucocidin PWS Peptone water sugar media QAL Quality Assessment Laboratory, DMRQC SLM Single Linkage Method (cluster analysis) SQL Structured query language (computer language) SS Salmonella-Shigella medium STAA Streptomycin Thallous Acetate agar medium TCH Thiophane carboxylic acid hydrazide TSI Triple sugar iron medium TTC 2,3,5-triphenyltetrazolium chloride UKNEQAS UK National External Quality Assessment Scheme

UPGMA VDU VP

Unweighted Pair Group Method by Averages (cluster analysis) Visual display unit Voges-Proskalier test

Codes used for various organisms Biogroup 3A Yersinia enterolitica biogroup 3A = Yersinia mollaretti Biogroup 3B Yersinia enterolitica biogroup 3B = Yersinia bercovieri B5W Acinetobacter calcoaceticus C27 Plesiomonas shigelloides CNW Campylobacter (catalase negative/weak) group Weeksella virosa Group Ilf Weeksella zoohelcum Group IIj Group IIk2 Sphingobacterium multivorum Group lib contains Flavobacterium indologenes Group IVe Alcaligenes-like organism (Oligella gen. nov. proposed to include also Moraxella urethralis) Group Vdl Achromobacter biotype 1 = Ochrobactrum anthropi Group Vd2 Achromobacter biotype 2 = Ochrobactrum anthropi Group Vel Chryseomonas luteola Group Ve2 Flavimonas oryzihabitans Vibrio fluvialis (also known as CDC group Group F EF6) Group JK Corynebacterium jeikeium Group M-4f Flavobacterium odoratum HB-1 Eikenella corrodens (Jackson & Goodman, 1972) 29911 group or biotype = Providencia alcalifaciens 32011 group or biotype = Haftiia alvei

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Index

Species names in brackets have either been transferred into another genus, fallen into disuse, or are in the process of being ratified by the Judicial Commission. abbreviations xviii, 266 aberrant forms 19, 54, 62, 140, 177 accession number 168, 169, 263 ACES buffer 163, 196, 266 acetoin, 28, 37, 41, 52, 53, 56-8, 73, 78; see also VP test acetoin-iodine decolorizer 214 acetylmethylcarbinol, 28, 37, 222; see also VP test Acholeplasma laidlawh 151, 164 Achromobacter 2, 95, 102, 109-12, 267 A. denitrificans 112 A. xylosoxidans (Type species) 107, 108, 111, 112 acid-alcohol decolorizer 214 Acidaminococcus 2, 94-6, 98 acid-fast bacteria 90-3 acid fastness 3, 25, 90 acid ferric chloride 219 acid mercuric chloride 34, 38, 85, 219, 228 acid production from sugars 30, 31, 51, 95, 172, 190, 198-200, 224 Acinetobacter 2, 4, 24, 26, 30, 95, 98, 101-5 (A. anitratus) 99, 101-4 A. baumanii 105 A. calcoaceticus (Type species) 99, 101-5, 239, 267 A. haemolyticus 105 A. johnsonii 105 A.junii 105 A. Iwoffii 99, 1 0 1 ^ acronyms 168, 263, 266 Actinobacillus 2, 95, 117-20, 176 (A. actinomycetemcomitans) 118—21, 153-6 A.equuli\\9, 120 A. lignieresii (Type species) 119, 120, 155 (A. rossi) 119 A.suis 119, 120 Actinobacillus from sow's vagina 119 Actinomadura 2, 51, 90 A. madurae (Type species) 90 Actinomyces 2, 51, 70, 75-82, 176 A. bovis (Type species) 79-81 A. denticolens 80 (A. eriksonii) SO, 81 A. hordeovulneris 80 A. howelli 80

A.israelii 12,80-2, 120

A. meyeri SO, 81 A. naeslundii 80, 81 A. odontolyticus 51, 80, 81 A. pyogenes 79-81 A. viscosus 80, 81 Actinomycetaceae 80

Actinomycetes 24, 50, 75, 79, 83 Adansonian concept, 3, 21, 179; see also numerical taxonomy (Aerobacter) 6, 129, 137, 167 aerobic spore formers 86-90 Aerococcus 2, 50-3, 58, 59, 61, 67, 175, 176 A. viridans (Type species) 27, 41, 50, 59, 67 Aeromonas 2, 95, 118, 121-3, 125-8, 176 A. caviae 127 (A.formicans) 126, 127 A. hydrophila (Type species) 126, 127, 239 (A. liquefaciens) 126, 127 A. media 127 (A.proteolytica), 126; see Vibrio proteolyticus A.punctata 126, 127 A. salmonicida 95, 118, 119, 123, 126, 176 A. salmonicida subsp. achromogenes 127 A. salmonicida subsp. masoucida 127 A. salmonicida subsp. salmonicida 127 A. shigelloides, 127, 128; see Plesiomonas shigelloides A. sobria 127 aerosols 27 aesculin hydrolysis 28, 58, 64, 190, 200, 223 media 73, 200, 223 agar9, 190, 192, 194,209 concentration 18, 26, 140, 191 Agrobacterium 2, 95, 109, 112 (A. radiobacter) 112 A. rhizogenes 112 A.rubi 112 A. tumefaciens (Type species) 111, 112 (Agrobacterium) yellow group 111, 112, 115 AIDS; Human Immunodeficiency Virus xviii aims of nomenclature and coding 169 air, ability to grow in 24, 26, 51, 95 Albert's stain 24, 214, 217 Alcalescens-Dispar (A-D) group, 135; see Escherichia Alcaligenes 2, 95, 104, 107, 267 A. aquamarinus 107 A. denitrificans 107, 108 A.faecalis (Type species) 21, 107, 108, 239 (A. odorans) 107, 108 A. piechaudii 107 (A. xylosoxidans) 107, 108 aldolase 30, 105 Allomonas 121 A. enterica (Type species) 121 alpha-haemolysis 35, 62 alpha-naphthol solution 220

317

INDEX Alteromonas 122 American National Standards Institute (ANSI) 249, 256, 266 American Type Culture Collection (ATCC) 239, 266 ammoniacal silver nitrate solution 214 ammonium oxalate-crystal violet stain 214 ammonium salt sugars (ASS) 31, 115, 198, 224, 266 amoebic dysentery 15 amylase in serum 10 anaerobes 83-6, 94-8 culture 16, 85 media for 7, 192,200 motility of 26, 86 anaerobically, ability to grow 16, 26, 95, 230 anaerobic cabinet 16, 83, 96, 212 anaerobic cocci, 51, 68, 94; see Acidaminococcus, Gemella, Megasphaera, Peptococcus, Peptostreptococcus, Veillonella anaerobic growth 17, 26, 51, 95 anaerobic jar 16, 26, 83, 212 anaerobiosis 16, 31, 68, 213 Anaerobiospirillum 2, 95, 150, 151, 158, 161 A. succiniciproducens (Type species) 151, 161 anaerogenic variants 135, 141, 177 Andrade's indicator 30, 188, 189 anthrax bacillus, 5; see Bacillus anthracis antibiogram 23, 263 antibiotic sensitivity xviii, 23, 24, 70, 97, 263 antibody, 23, 54; see also monoclonal antibody anticoagulant 17, 32, 221 antigen 62-6, 73, 114, 145, 207 polysaccharide 62 antiserum 62, 63 API system 20, 42, 89, 172, 183, 266 approaches to bacterial systematics xiii, 1, 173, 174, 260-2 approved lists of bacterial names xiii, xviii, 4-6, 170, 174, 252-9 aqueous solutions for staining 214 Arachnia 2, 51,15-82, 176 A. propionica (Type species) 76-82 Arcanobacterium 2, 51, 70, 75, 79 A. haemolyticum (Type species) 75-7, 79 Arcobacter2, 150, 151, 158, 161 A. cryaerophilus 158, 161 A. nitrofigilis (Type species) 161 arginine hydrolysis 32, 53, 115, 223, 227 media 200-2 arithmetical summation 173 (Arizona)', see Salmonella arizonae Arthrobacter 4, 55, 57, 70 Asakusa group, 132, 133; see Edwardsiella ascitic fluid 11,30 asporogenous 25, 49, 76 assay, media for 9 autoclaving 11-14, 187 inspissation 14 momentary autoclaving, 13, 14; see also HTST relation between temperature and pressure 13 automated methods 44, 45 Avium-intracellulare-scrofulaceum group; see Mycobacterium B5W group, 104, 267; see Acinetobacter Bacillus 2, 49, 51, 86-90 asporogenous strains 49

318

B. alvei 88 B. amyloliquefaciens 88, 89, 90 B. anthracis 86-9, 201, 237 (B. anthracoides); see B. cereus B. badius 88 B. brevis 88 B. cereus 86-9 B. circulans 88 B. coagulans 87, 88 B.firmusSS, 90 B. laterosporus 88 B. lentus 88, 90 B. licheniformis 87, 88, 89 B. macerans 88 B. megaterium 88 B. mycoides 87-9 B.pallidusSl B. pantothenticus 88 B. poly myxa 88 B.pumilus 88 B. sphaericus 88 B. stearothermophilus 13, 87, 88, 239 B. subtilis (Type species) 12, 87, 88, 89, 239 (B. thermodenitrificans) 87 B. thuringiensis 87-9 bacitracin sensitivity 54, 62, 241 bacteria Gram—negative 94—164 Gram-positive 50—93 bacterial identification profile (BIP) 183 bacteriocin 17, 114 Bacteriological Code of Nomenclature xii, xiii, 4-6, 252, 253 (Bacterium) 129 Bacterionema 70, 71 Bacteroides 2, 94-8 B. asaccharolyticus 96, 97 B. bivius 96, 97 B. capillosus 96 (B. corrodens) 96, 157, 158 B. disiens 96, 97 B. distasonis 96 B.fragilis (Type species) 96, 97 B. intermedius 96 B. melaninogenicus 96, 97 B. oralis 96, 97 B. ovatus 96 B ruminocola 96 B. splanchnicus 96, 97 B. thetaiotaomicron 96, 97 B. uniformis 96 B. ureolyticus 96, 97 B. vulgatus 96 Ballerup-Bethesda group, 131, 133; see Citrobacter freundii barred diphtheroids 72 Bartholomew group, 132, 133; see Edwardsiella base composition of DNA 1, 68, 79, 100, 101, 108, 123 BASIC computer language 249, 266 BCG 93, 266 BCYE medium 162, 163, 196, 266 beetles 191 (Beneckea); see Vibrio 121 Benedict's solution 219 Bergey's Manual of Determinative Bacteriology 1, 48, 254 Bergey's Manual of Systematic Bacteriology 3, 48, 167

INDEX (Betabacterium), 75; see Lactobacillus beta-galactosidase 39, 65, 224, 233 beta-toxin (staphylococcal) 29, 56, 63 Bifidobacterium 2, 51, 79-83, 176 B. bifidum (Type species) 83 bile 11 media 196, 223 salts 11,209,211 solubility 29, 223 tolerance 223 biotypes 1, 143, 152 Bismark brown 214 bite infections 80, 118, 161 biurettest 193,210 bleeding polenta 21 blood culture 17,72 haemolysis of 34, 35, 63, 73, 85, 194, 195 medium constituent 10, 194-6, 206 peptic digest 195 specimens 17 Bordet-Gengou medium 106, 196 Bordetella 2, 95, 102, 103, 106-8, 176 B. bronchiseptica 21, 95, 106, 108, 239 B.parapertussis 95, 103, 106 B. pertussis (Type species) 95, 103, 106-8 botulism 83 Boyd's dysentery bacilli; see Shigella boydii Branhamella 2, 95, 98, 101 B. catarrhalis (Type species) 101 (B. caviae); see Neisseria (B. cuniculi)', see Neisseria (B. ovis); see Neisseria Brazilian purpuric fever, 153; see Haemophilus Brevibacterium 70, 71 brilliant green-containing media 141 Brochothrix 2, 51, 69, 70, 73-6 B. campestris 13,74 B. thermosphacta (Type species) 73-5 bromcresol purple 30, 189 bromthymol blue 30, 189 toxicity of 28 bromthymol blue salt teepol (BTBST) agar medium (for Vibrio) 122, 196 broth-based sugar media 198, 199, 224 Browne's tubes 188 Brucella 2, 95, 102, 103, 105, 106, 176 B. abortus 103, 105, 106 B. canis 106 B. melitensis (Type species) 103, 105, 106 B. neotomae 106 B. ovis 106 B. suis 103, 105, 106 Budvicia 2, 148 B. aquatica (Type species) 148 buffer solutions 222 Buffered Single Substrate (BSS) tests 29, 109, 115, 266 Buttiauxella 2, 95, 128, 131, 133 B. agrestis (Type species) 130, 131, 133, 239 CAMP test 29, 63, 73, 118, 224 Campylobacter 2, 95, 121, 150, 151, 158-60, 226, 266, 267 C. coli 158-60 C.concisus 158, 160

(C. cryaerophila) 158, 161; see Arcobacter C. curvus 160 C. fetus (Type species) 158-60, 239 C. hyointestinalis 158, 159 C. jejuni 158-60,239 C. lari 158, 159 (C.pylori{disJ), 158, 160; see Helicobacter pylori C. rectus 160 C. sputorum 158, 160 C. upsaliensis 158-60 candle jar 26 capillary tube test for motility 26 capnophilic; see carboxyphilic capsule stains 217 carbohydrate 11, 30, 31, 95, 189, 190, 210 acid from 224 breakdown 3, 30, 31,224 fermentation, 11, 27, 109, 224 media 11, 12, 30, 31, 198-200 oxidation 3, 27, 31, 109, 224 sterilization 11, 198-200 utilization 30, 31 carbol fuchsin stain 214 carbon dioxide CO2 effect 17, 118, 152, 161 growth in presence of 60, 61, 63, 79 increased concentration 16, 17, 26, 79, 100, 152 inhibition by 17 carbon source utilization (CSU) 12, 31, 201, 266 organic acids 31, 201, 225 carboxyphilic 263 Cardiobacterium 2, 95, 117-20 C. hominis (Type species) 119, 120 cards, punched xviii, 47, 175-8, 243-8 aggregation of cards 176 notching of master cards 175, 243, 244, 247 probing of cards 243, 244, 248 Carnobacterium, 76; see Lactobacillus C. divergens 76 C. piscicola 76 carry-over of nutrients 31, 34, 151 casein hydrolysis 33, 227 media 38, 201 catalase 24, 26, 27, 51, 95, 225, 226, 241 semiquantitative test 27, 91, 226 category 253, 261,263 Cedecea 2,95, 128, 130, 131, 133 C. davisae (Type species) 131, 133 C.lapageim, 133 C.neterim, 133 cell wall 3, 52, 62, 171 centroid, 181; ^^^ also identification statistics Cesares-Gill flagella stain 218 chancre 15 characterization 4, 5, 18, 21-^5 tests for 24-42, 182, 219-238 characters 3 , 2 1 ^ 2 anomalous 49 choice of 22 Gram-negative bacteria 4, 94—164 Gram-positive bacteria 4, 50-93 media, effect of 22 primary 24

319

INDEX secondary 28 specific 3 chemotaxonomy xviii, 52, 179, 263 chitin, media containing 31, 226 chitinolytic activity 31 chlororaphin 113 chlorphenol red 189 chocolate agar 17, 152, 195, 212 chocolate tellurite agar 72, 196 Christensen's citrate medium 31, 201 Christensen's urea medium 41, 115, 208, 238 chromatography 69, 87, 92, 100 Chromobacterium 2, 95, 112, 123, 128 (C. lividum), 112; see Janthinobacterium C. violaceum (Type species) 112, 113, 121-3, 128, 176 Chryseomonas luteola 2, 95, 111, 116, 267 chrysoidin 214 citrate buffer 222 media 9, 201 use of as carbon source 31, 212, 225 Citrobacter 2, 95, 128, 130-2 C. amalonaticus 131, 133 C.diversus 131, 133 C. freundii (Type species) 131-3 (C. intermedius) 131 C.koserim, 133 classification 1, 3-5, 21, 165-8 all purpose 166, 167 computer, 167, 168; see numerical taxonomy convenience 167 disciplinary 166 hierarchical 4, 166, 167 minimal difference 166 statistical 167, 168 Clavibacter 70; see Corynebacterium cleaning glassware 188 CLED (cystine-lactose-electrolyte deficient) medium 140, 266 Clostridium 2, 51,83-6 culture 18, 84 isolation 18, 84 C. barati 83-5 C. bifermentans 42, 84-6 C. botulinum 83-5 C. butyricum (Type species) 84-6 C.carnis 51, 84 C.chauvoei83, 84 C. difficile 84, 86 C.fallax84, 85 C. histolyticum5\, 84 C. innocuum 84, 85 C. novyi 84,85 (C. oedematiens) 84 C. perfringens 76, 83-5, 176, 239 C.putrefaciens 83, 84 C. ramosum 83, 84 C. septicum 84 C. sordelli 42, 84, 85 C. sphenoides 84, 85 C. sporogenes 83-5, 239 C. subterminale 83, 84 C. tertium 51, 84 C. tetani 83-5, 239 (C. welchii); see C. perfringens

320

clot formation 32, 226 CNW group, 160, 267; see Campylobacter coagulase 3, 10, 21, 31, 32, 54, 56, 226, 241 COBOL computer language 249, 266 coccal transformation 227 cockroaches, 135; see Escherichia blattae codes aims of 169 bacteriological xiii, 4-6, 168-72 descriptive 169 numerical 169 colicin 145 collection, strain 179, 258 (Colloides), 104; see Acinetobacter colony morphology 22, 23 colony, mixed 17, 18,48 Comamonas 109 C. terrigena (Type species) 109 computers bacterial identification 21, 179-83, 250 classification 167, 168 database 179, 180,250 disks 250 operating system 250 programming 249-51, 257 use of 249-51 conserved name 253, 257, 263 contamination 4, 73, 164, 166, 185, 212 of cultures 18, 19 control laboratory quality 184-6, 208 media 208-13 sterilization 212 cooked meat media 8, 186, 192 Corynebacterium 2,51, 69-72 (C. acnes), 70; see Propionibacterium C. ammoniagenes 70, 71 C. amycolatum 11 C.bovisll,12 C.cystitidislO,ll C. diphtheriae (Type species) 5, 69-72 gravis type 71,72 intermedius type 71, 72 mitis type 11,12 (C. equi), 29, 70; see Rhodococcus (C. haemolyticum) 70; see Arcanobacterium (C. hoagii) 70; see Rhodococcus C. kutscheri; (C. murium) 71, 72 C.jeikeium 11,12,261 C. matruchotii 70-2 C. minutissimum 70, 71 C. mycetoides 70, 71 C. pilosum 70, 71 C. pseudodiphtheriticum 11 (C. pseudotuberculosis); (C. ovis) 29, 71, 72 (C. pyogenes); see Actinomyces 70 C.renalelO, 71 C. striatum 11 (C.ulcerans) 71,72 C. vaginale 156 C. xerosis 71, 72 coryneform bacteria 75 coryneform-actinomycete intermediate group 75-9 Craigie's method for motility 26

INDEX creatine solution 219 cresolred 189 cultures 15-20 contaminated 17-19, 48 disposal of 12, 187 pure 17-20 storage 185, 186,242 test 239^1 Curtobacterium, 70; see Corynebacterium CYLG (casein, yeast, lactate, glucose) broth 193, 266 cytochromic oxidase, 27, 53, 233; see also oxidase cytophagas 24, 25 D, d, (d) characters xvii, 46, 106, 176 dairy organisms; see Lactococcus, Lactobacillus, Leuconostoc, Streptococcus dangerous pathogens 187 Data Protection Act 251 deamidases 29 deaminases 234 decarboxylase media 202 tests 32,115, 227 decolorization of indicator 31 decolorization of stained bacteria 68, 100, 102 decolorizer(s) 92 Deleya 107; see Alcaligenes dendrogram, 173, 263; see also numerical taxonomy denitrification 32, 38, 39 de(s)oxycholate medium (DCA) 16, 18, 141, 146, 266 deoxyribonuclease (DNase) 32, 33, 56, 202, 228, 241 Dermobacter 70 description of an organism 170, 252, 253, 263 Determinator21, 172 dextran production 60, 61, 68, 235 media 200 dextrin 11 diagnosis 263 dichotomous (diagnostic) key 172 digest broth 192, 193 digestion casein 33, 227 egg 33, 228 meat 33, 228 serum 33, 228 diphtheroids 69-75 (Diplococcus), 62; see Streptococcus pneumoniae direct microscopy 15 disk operating system (DOS) 250 disposal of cultures 12, 187 division of bacteria into major groups 261 DNA base composition 1, 68, 69, 79, 100, 101, 108, 123, 125 DNA homology 1, 52, 87, 106, 179, 261 DNA probe 1, 44, 123, 173, 265 Dorset egg medium 8, 14, 194 double-sugar media 20 D r 263 Duguid's method for showing capsules 217 Durham tubes 68, 198 dysentery amoebic 15 bacillary 15 (Eberthella) 167, 169

Edwardsiella 2, 95, 128, 132, 133 (E. anguillimortifera) 132, 133 E. tarda {Type species) 130, 132, 133, 240 effective publication 263 egg, digestion of 33 egg media 7, 92, 194,204 egg-yolk reactions 18, 36, 37, 54, 85, 204 Ehrlich's reagent 219, 230 Eijkmantest 150,228 Eikenella 2, 95, 157-9 E. corrodens (Type species) 96, 97, 151, 154, 157-9, 164, 267 electrophoresis 65, 87 eliminating tests 19 eltor biotype of Vibrio cholerae 125 EMB (eosin-methylene blue) agar 16 Entamoeba 15 E. coli 15 E. histolytica 15 Enterobacter 2, 6, 95, 128, 1 3 2 ^ E.aerogenes 132^,239 E. agglomerans, 130, 133, 134; see Erwinia herbicola E. cloacae (Type species) 1 3 2 ^ , 239, 240 E. gergoviae 132, 133 (E. liquefaciens); see Serratia liquefaciens E.sakazakii 132, 133 E. taylorae 132 enterobacteria, Enterobacteriaceae xi, xviii, 1, 95, 128-50, 176 enterococci 62, 66, 67, 78 Enterococcus 2, 51, 59, 61, 65-7, 176 E. avium 61 E. casseliflavus 61, 67 E. durans 61 E.faecalis (Type species) 61, 66, 67, 239 E. faecium 61, 66 E. gallinarum 61, 67 E. mundtii 61 enterotoxins clostridial 83 staphylococcal 54, 56 vibrio 123, 124 enzymes constitutive 20, 42, 43 formed by bacteria 29, 38, 54, 65, 83, 237 induced 20, 34, 39 in serum 10 epithet, specific 3, 6, 78, 258 error, test 22, 168, 180 Erwinia2,95, 128, 134 E. amylovora (Type species) 134 E. herbicola 130, 133, 134, 149 Erysipelothrix 2, 51, 72, 75-8, 176 E. rhusiopathiae (Type species) 75, 77, 78, 81 E. tonsillarum 78 Escherichia 2, 95, 128, 130, 135, 136 anaerogenic biotypes 135 (E. adecarboxylata), 135, 136; see Leclercia E.blattae 135 E. coli (Type species) 135, 136, 149, 239 E.fergusonii 135, 136 E. hermanii 135, 136 E. vulneris 135, 136 ethylhydrocuprein (EHC; optochin) 33, 228 Eubacterium 2, 51, 83, 84, 86, 176

321

INDEX E. lentum 86 E. limosum (Type species) 86 Ewingella 2, 149 E. americana (Type species) 149 extracts for grouping streptococci 23, 62 Falkow's decarboxylase tests 202, 227 family 1, 122, 167, 253, 261, 263 fastidious organisms 19, 33, 150 media for 8 FBP (Ferric Bisulphite Pyruvate) medium 202 fermentation 3, 10, 94; see also carbohydrates heterofermentative 75 homofermentative 75 tests 3, 27, 30, 224, 225 ferrous chloride gelatin medium 202 fibrinolysin 32, 56 Fildes' digest agar 84, 195 Fildes' peptic digest 195 filters, bacterial 14, 190 filtration 189, 190 fimbria 3 flagellum (-a) 3, 24 arrangement 3 stain 24, 215, 218 Flavescens group; see Mycobacterium Flavimonas coryzihabitans 2, 95, 111, 167, 267 Flavobacterium 2, 95, 109, 116, 117 Group lib, 116, 117; see F. indologenes Group Ilf, 116, 117; see Weeksella virosa Group IIj, 116, 117; see Weeksella zoohelcum F. aquatile (Type species) 116 F. breve 117 F.gleum 116, 117 F. indologenes 116, 117, 267 F. meningosepticum 116, 117 F. multivorum, 116, 117; see {Sphingobacterium) F. odoratum 117 F. spiritovorum, 116, 117; see {Sphingobacterium) F. thalpophilum 117 flow chart 46 fluorescence-denitrification (FN) medium 32, 203, 266 fluorescent antibody (FA) 23, 106 fluorescent microscopy 23 fluorescin 113 (Fluoribacter); see Legionella food poisoning 83, 86, 122, 125, 128 FORTRAN computer language 249 Fortuitum group; see Mycobacterium fractional sterilization 14 Francisellal, 117-21 F. novicida 120 (F. philomiragia) 121 F. tularensis (Type species) 118, 120, 121 Frazier's reagent 219 test 34, 228 freeze-drying 185 Friedlander's bacillus; see Klebsiella pneumoniae frozen cultures, preservation of 185, 186, 242 Fusobacterium 2, 94-8 F. mortiferum 98 F. necrophorum 97, 98 F. nucleatum (Type species) 97, 98 F. varium 98

322

G+C% content or ratio, 264; see DNA base composition (Gajfkya) 61, 61 Gardnerella 2, 150, 151, 154-7 G. vaginalis (Type species) 71, 151, 154-7 gas production 30, 76, 146, 172 methods of detection 198, 224, 225 gelatin 11, 189, 210 hydrolysis 33, 34, 177, 228 media 203 stab cultures 23, 33 Gemella 24, 49, 51, 59, 68, 94, 102, 175-6 G. haemolysans (Type species) 61, 68, 99, 102 genera 1-6, 50, 129,261,264 GENMAT computer program 250 genotype 264, 265 geographical variants 92, 104, 180 glanders 113; see Pseudomonas mallei glassware cleaning 188 sterilization 188 glossary xviii, 263 gluconate 34 medium 203 test 229 glucose 95, 224 breakdown 31 media 195, 197, 203, 223 glutamic acid decarboxylase 227 glycerol agar 195 glycine buffer 203 gonococcus; see Neisseria gonorrhoeae Gordonae group; see Mycobacterium Gram's staining method 3, 24, 94 Gram reaction 3, 15, 24 Gram negative bacteria 23, 24, 94, 95 Gram positive bacteria 2-4, 24, 50, 51, 94 Gram variable bacteria 4, 86, 100 group antigen(s) 62 group D streptococci; see Enterococcus group E streptococci from swine; see Streptococcus porcinus groups and divisions 50, 94, 264 convenience 50, 167 major 50, 94 phylogenetic 1, 165, 173 serological 62 growth 34, 212, 229 growth inhibition 36 growth under special conditions 'X' and ' V factor requirements 238 with increased NaCl 229, 236 at pH 9.6 66 at5°C 121,237 at44°C 105,228 haemolysis 34, 35, 85, 241 Haemophilus 2, 95, 120, 150-6 growth factors, 'X' and ' V 153-6 isolation 150 (H. actinomycetemcomitans) 120; see Actinobacillus H. aegyptius 153 (H. agni) 155 H.aphrophilus 118, 119, 121, 152 H.avium 152, 154, 155

INDEX Haemophilus (cont.) H.ducreyi 152, 154, 155 (H. equigenitalis) 155 H. gallinarum 152, 155 H. haemoglobinophilus 154 H. haemolyticus 152, 154 H. influenzae (Type species) 152-6 H. paracuniculus 155 H. paragallinarum 152, 154, 155 H. parahaemolyticus 152, 154 H. parainfluenzae 152, 154 H. paraphrohaemolyticus 152, 154 //. paraphrophilus 119, 152, 154, 155 H.parasuis 152, 154, 155 H. pleuropneumoniae 155 H.segnis 152, 154, 155 (7/. somnus) 155 (//. vaginalis), 154-7; see Gardnerella Hafnia 2, 95, 128, 134-7 //. tf/ve/ (Type species) 130, 136, 137, 149, 267 halophilic organisms 31, 122, 229, 264 heat, sterilization by 12-14 Helicobacter 2, 95, 150, 151, 158, 160 //. ci/wtt/i 158, 160 //./«?/« 160 H.fennelliae 158, 160 //. mustelae 160 //. pjy/on (Type species) 158, 160, 239 (Herellea); see Acinetobacter Hikojima antigenic subtype, 125; see Vibrio hippurate hydrolysis 35, 64, 229 medium 203, 212 homology, DNA 1, 52, 87, 106, 179, 261, 264 hot air sterilization 13 Hugh & Leifson (OF) medium 198 test, 180, 224; see oxidation-fermentation (OF) test hybridization xviii, 5, 6, 63, 135, 171, 264 hydrocele fluid 11 hydrogen peroxide 219, 230 hydrogen sulphide (H2S) 22, 35, 36, 49, 230 hydrolysis aesculin 28, 200, 223 arginine 32, 223, 227 gelatin 33, 177, 228 salicin212 starch 212 sugars 10 Tween 156, 157, 238 urea 41, 238 hygiene, laboratory 187 hyperspace, hypersphere, 181, 264; see also identification statistics Identicards 175-8, 243-8 minimal difference cards 175 identification 1,46-9, 171-3 arithmetical systems 173 by computer 48, 179-83, 250 databases 179-82,250 keys 172 matrices 179, 180 mechanical systems 21, 22, 44,45, 172 practice of 46, 48,49

principles 181, 182 profiles, 182, 266; see Bacterial Identification Profiles punched cards 172, 175-8, 243-8 statistical 181, 182 tables xvii, 46-9, 171, 179 theory 46-8 identification statistics 181, 182 likelihood 181, 182 modal likelihood 182 most typical 182 normalization 182 probabilistic 181 taxon-radius model 181 identification systems agar based 43 dehydrated substrates 42, 43 kits 20, 42-4 manual 42 mechanized 44, 45 illegitimate name 264 IMViC reactions 34, 135, 266 Inaba antigenic subtype, 125; see Vibrio inactivation of enzymes 10 incertae sedis 264 indiaink215, 217 indicators (pH) 12, 188, 189 decolorization 31 indole 3, 36, 209, 230 infected containers and material, sterilization of 12, 187 infusion broth 193 inhibition of growth 36 inoculum size 180 insertion site (of flagella) 24 inspissation 14 interaction between products 20 iodine, Lugol's 2, 5, 220 iridescence 73 isolation of bacteria 15-20 Janthinobacterium 2, 95, 109, 112, 113, 123 / . lividum (Type species) 111-13, 123, 128 Jonesia 72 / . denitrificans (Type species) 72 Kanagawa test for Vibrio parahaemolyticus 125 KCN medium 12,204 test 36, 231 key dichotomous 46, 172 numerical diagnostic 172 KIA (Kligler iron agar) 20, 266 Kingella 2, 95, 103-5 K. denitrificans 105 K. indologenes 105 K. kingae {Type species) 103, 105 King's media 114, 197 Kirkpatrick's fixative 215, 218 kits, identification 20, 42^4 Klebsiella 2, 6, 95, 128, 130, 136-8 pathogenicity 137 (K. aerogenes) 136, 137 (K.atlantae) 137 (K.edwardsii) 137

323

INDEX Klebsiella (cont.) K.oxytoca 136, 138 K. ozaenae 136 K.planticola 138 K. pneumoniae (Type species) 136-8 K.pneumoniae subsp. aerogenes 136, 137, 239 K. pneumoniae subsp. ozaenae 136, 137 K. pneumoniae subsp. pneumoniae 136, 137 K. pneumoniae subsp. rhinoscleromatis 136, 137 K. rhinoscleromatis 136 K. trevisanii 138 Kluyvera 2, 95, 128, 130, 131, 136, 138 K. ascorbata (Type species) 138 K. cryocrescens 138

Knoxplate9, 19

Koserella 2, 149 K. trabulsii (Type species) 149, 150 Koser's citrate medium 31, 201, 209 Kovacs' indole reagent 219, 231 Kurthia 2, 51,69,70,74-6 K. gibsonii 74, 75 K. zopfi (Type species) 74, 75 L (Lister) forms 164 Lactobacillus 2, 51, 75-8, 176 catalase positive 26, 76 medium 199, 205, 224 motile 76 L. acidophilus 11, 78 (L. bifidus), 83; see Bifidobacterium L. brevis 11 (L. carnis), 76; see Carnobacterium L. casei 76, 77 (L. cellobiosus); see L.fermentum L. confusus 68 L. delbrueckii 11, 78 (L. divergens), 76; see Carnobacterium L.fermentum 11 L. gassed 11 L. jensenii 11 (L. leichmanii); see L. delbrueckii (L. piscicola), 76; see Carnobacterium L. plantarum 11, 78 L. salivarius 11, 78 L. viridescens 68 Lactococcus, 2, 51, 59, 62, 66; see Streptococcus L. lactis (Type species) 61, 62, 66 L. thermophilus 62 lactose fermentation 18, 19, 30, 39, 177, 201, 225 lactose (10%) media 30, 39 Lancefield serology, streptococci 61—6 language, computer 249, 250 layered plates 192 lead acetate papers 22, 35, 221 lecithinase 18,36,37,85 lecithovitellin (LV) test 36, 37, 231, 267 medium for 204 Leclercia adecarboxylata (Type species) 135 Legionella 2, 95, 150, 151, 161-3 gas-chromatography 163 isolation 163 L. anisa 162 L. birminghamensis 162, 163 L. bozemanii 162

324

L. brunensis 163 L. cherrii 162 L.dumoffii 162 L. erythra 162 L.feelii 162 L. gormanii 162 L. gratiana 163 L. hackeliae 162 L. israelensis 162 L. jamestowniensis 162 L.jordanis 162, 163 L. longbeachae 162, 163 L. maceachernii 162 L. micdadei; (Tatlockia) 162, 163 L. moravica 163 L. oakridgensis 162 L. parisiensis 162 L. pneumophila (Type species) 162, 163, 239 L. quinlivanii 163 L. rubrilucens 162 L. sainthelensis 162 L. santicrusis 162 L. spiritensis 162 L. steigerwaltii 162 L. tucsonensis 162, 163 L. wadsworthii 162 legitimate name 6, 264 Leminorella 2, 149 L. grimontii (Type species) 149 L. richardii 149 leprosy 15, 16, 92 leptospiras 4 Leptotrichia, 2, 95; see Fusobacterium L. buccalis (Type species) 97 leucocidin, staphylococcal 56 Leuconostoc 2, 51, 59, 61, 67, 68, 176, 224 L. mesenteroides (Type species) 68 levan production 60, 61, 68, 235 media for 200 (Levinea), 130, 131, 133; see Citrobacter (L. amalonatica) 131 lipolytic activity 41 liquoid (anticoagulant) 16, 17, 161,221 (Listerella), 70, 72; see Listeria Listeria 2, 51, 69, 70, 72-8 (X. denitrificans), 72; see Jonesia L. grayii 72, 74 L. innocua 73, 74 L. ivanovii 73, 74 L. monocytogenes (Type species) 72-4, 77, 78 L. murrayi 72, 74 L. seeligeri 73, 74 L. welshimeri 73, 74 Listonella 121, 122 Loeffler's methylene blue stain 24, 215 Loeffler's serum medium 71, 204 (Loefflerella), 111; see Pseudomonas Lowenstein-Jensen medium 17, 204 Lucibacterium, 121; see Vibrio Lugol's iodine 215, 220 lysine decarboxylase 32, 115, 202, 227 lysostaphin 54 lysozyme 54, 263 lytic enzyme for streptococcal grouping 220

INDEX MacConkey's media 146, 197, 199, 213 Mcllvaine's buffer 222 maintenance of test cultures 8, 185, 186, 239 major groups of bacteria 50, 94, 262 malachite green 25, 85, 92, 214 malaria parasites 15 malonate medium 205 test 37, 231 maltase in serum 10 mannitol yeast-extract (MYE) agar 197 master cards for identification 175, 243 meat 10 digestion of 33 extract of 10, 209 infusion 10, 80, 194 media 7-9, 188-213 basal (non-nutrient) 9, 194 basic (nutrient) 1 9 2 ^ carbohydrate 11, 198-200 characterization 9 clarification of 189, 190, 207 commercially available 7, 42, 44 constituents 9-12, 79 control 208-13 defined 7, 12 dehydrated 7, 42 differential 18, 195-7,213 enriched 33, 194-5 enrichment8, 141, 146, 194, 195 faulty 210, 213 inhibitory 8, 16-19, 146 isolation 8, 16, 17 maintenance 8, 185 microbiological assay 9 multitest 19, 42 non-inhibitory 16-18 nutritional requirements 8 pigment production, for 197, 198 quality of xix, 7, 184, 208 raw materials for 209 reduced (PRAS) 7, 8, 267 screening 9 selective 8, 16-19, 48, 195-7, 213 solid 8, 31, 58, 82, 213 sterility tests 212 sterilization 7, 12, 189 storage of 189, 190 sugar 19,27, 30, 198-200 test organisms 239 volumes 191 Megasphaera, 2, 94-6, 98; see also anaerobic cocci melioidosis 113 meningitis 17, 64 meningococcus; see Neisseria meningitidis mesophil(e) 92, 264 metabolic fatty acids 37, 52, 62, 163, 171 metachromatic granules 24, 72, 217 methylene blue stain 12,71,215 methyl red (MR) 267 indicator 189 media for MR test 199 MR test 37, 231

solution 220 Microbacterium, 70; see Corynebacterium Micrococcus 2, 51-54, 57, 58 M. agilis 58 M. kristinae 57, 58 M. luteus (Type species) 57, 58 M. lylae 58 M. nishinomiyaensis 57, 58 M. roseus 57, 58 M. sedentarius 58 M. varians 57, 58 micromethods 8, 20, 4 2 ^ microscopy 15, 23, 24, 85 direct 15 electron 3, 24 phase contrast 24, 25, 87, 114 milk as medium 38, 201, 205, 231 (Mima) 98, 102; see Acinetobacter minidefinitions, limitations of 76, 86 minute haemolytic streptococci 60, 64 mites 191 Mobiluncus 2, 94 modal likelihood 182 Moellerella 2, 149 M. wisconsensis (Type species) 149 Moeller's spore staining method 25, 217 Molisch's test for carbohydrate 211 momentary autoclaving 13, 14 monoclonal antibody 44, 166 Moraxella 2, 95, 102-5, 176 M. bovis 102, 103 (M. equi), 102, 103 (M. kingii); see Kingella kingae M. lacunata (Type species) 102, 103, 104 (M. Iwoffii); see Acinetobacter M. nonliquefaciens 103 M. osloensis 103 M. phenylpyruvica 103, 104 M. urethralis 103, 104, 267 Moraxella-Acinetobacter group 102-7 mordants 215, 216 Morganella 2, 5, 95, 128, 138 M. morganii (Type species) 130, 136, 139, 239 morphology 4, 22, 24 morphological groups 24 relation to growth phase 24 most typical likelihood 182 motile and non-motile variants 49, 75 motility 26, 73, 95, 222, 232, 241 gliding 24, 26 medium 12,205 MR test; see methyl red Mueller Hinton medium 194 Muir's capsule staining method 217 mordant 215 multitest, media 19, 20 MUMPS computer language 249, 267 Mycobacterium 2, 51, 90-3, 176 Avium-intracellulare-scrofulaceum group 91 Flavescens group 91 Fortuitum group 91 Gordonae group 91 Scotochromogenic psychrophilic group 91 Smegmatis-phlei group 91

325

INDEX Mycobacterium (cont.) Terrae group 91 (M. aquae)91 (M. avium)9\ (M.balnei)9\ (M. borstelense) 91

M.bovis 17,91,92

M. chelonae 91,92 M.fortuitum 90,91,239 M.johnei 17 M.kansasii91,239 M.leprae 16,92 M. lepraemurium 92 M. marinum 91,93 M. microti 92 (M. minettii) 91 M. paratuberculosis 92 (M. perigrinum) 91

M./7/*/e/17,92,93,239

M. smegmatis 239 M. tuberculosis (Type species) 91-3, 239 M. ulcerans 91 M. xenopi 91 mycolic acids 70, 90 mycoplasmas 4, 16, 19, 163 Mycoplasma 95, 150, 151, 163, 164 NAG or 'non-agglutinable vibrio' 125, 267 Nagler reaction 37 name 5, 6, 168,260 appropriate 6 approved 6, 252-9 common 5 epithet 6, 258, 259 illegitimate 264 legitimate 6, 264 scientific (latinized) 5, 169, 252, 253 vernacular 5, 169,252,261 National Collection of Type Cultures xi, 25, 47, 100, 179, 180, 239,256,267 NCV or 'non-cholera vibrio' 125, 267 (Necromonas), 121, 126, 127; see Aeromonas needling, of punch cards 243, 244, 248 negative stain for capsules 217 Neisser's stain 24, 215, 218 Neisseria 2, 95, 9%-\0\ false neisseriae 100 N. animalis 101 N. canis 101 (TV. catarrhalis) 99-101; see Branhamella N. caviae 100 N.cinerea 99-101 N. cuniculi 100 N. denitrificans 101 N.elongata 99-101 N.flavescens 99-101 N. gonorrhoeae (Type species) 99, 101 N. lactamica; (TV. lactamicus) 99, 101 N. meningitidis 99 N.mucosa 99, 101 N. ovis 100 (N. pharyngis) 101 N.subflava 99, 101 (N.flava) 99, 101

326

(TV. perflava) 99, 101

(N.sicca) 99, 101

neotype 170, 253, 257, 258, 264 Nessler's reagent 220 neutral red 189 niacin (nicotinic acid) production 38 reagents for test 220 test 232 nitrate media containing 206 test for residual 38, 107, 211 nitrate reduction 38, 241 interpretation of tests 39 tests for 211,232 nitrite media containing 206 reagents for test 220 reduction of 38, 39 test for 38, 211,233 nitrogen amino 211 fixation 138 nutrition 39 total 210 Nocardia2,5\,9Q,9\, 176 N. asteroides (Type species) 90, 91 N. brasiliensis 90, 239 (TV. madurae); see Actinomadura madurae N. otitidis-caviarum 90, 239 nomenclature xviii, 1,4, 168-71, 260, 261 aims 169 Code of 170, 171,252,253 pronunciation 169 nomen periculosom 174, 261, 264 'non-fermenting' bacteria 18, 30, 107, 213 non-saccharolytic bacteria 107-9 normalization 182 notching of punched cards 175-8, 244 cards for unknowns 178 master cards for taxa 175, 177, 244-8 numerical diagnostic key 129, 172 numerical taxonomy 148, 167 cluster analysis 168 dendrograms 168 ordination 168 principal components analysis 168 similarity matrix 168 systematics xiii, 1, 173, 265 numericlature 169 nutrient media 192, 194, 199 nutritional requirements 8, 87, 115 O/129 (pteridine reagent), sensitivity to 24, 39, 233 Obesumbacterium 2, 149 O. proteus (Type species) 149 (Oceanomonas alginolytica); see Vibrio Ochrobactrum 2,95, 111 O. anthropi (Type species) 111, 112, 267 OF test; see oxidation-fermentation test Ogawa antigenic subtype, 125; see Vibrio (Oligella), 104, 267; see Moraxella OMNI-serum, for pneumococci 64 ONPG

INDEX medium 206 test 39, 233 operational taxonomic unit (OTU) 265, 267 optochin (ethylhydrocuprein) 33, 62, 65, 228, 241 order (category or rank) 1, 253, 255 ordination; see numerical taxonomy organic acids as carbon sources 31, 201 ornithine decarboxylase 32, 115, 202, 227 oven, sterilization in hot air 188 oxalic acid paper, test for indole 221 oxidase24, 27, 51,95, 241 reagents 220 test 233 oxidation-fermentation (OF) test 3, 27, 180, 220, 241 pancreatic extract 193, 210 (Pantoea agglomerans); see Erwinia herbicola paper filter 190 test 19,221 (Paracolobacterum anguillimortiferum), 132; see Edwardsiella parapertussis; see Bordetella Pasteurella 2,95, 117-9, 176 (P.avium) 155 P. gallinarum 119 P. haemolytica type A 118, 119 P. haemolytica type T 118, 119 P. multocida (Type species) 118, 119 P. pneumotropica 118, 119 (P. pestis) 118, 119; see Yersinia pestis

P.ureae 118, 119

(P. volantium) 155 Pediococcus 2, 51, 59, 61, 67, 68, 176 P. damnosus (Type species) 68 (P. homari) 67 P. pentosaceus 67 (P. urinae-equi) 67 penicillin, discovery of 19 pentoses, detection of 211 Peptococcus 2, 51, 59, 68, 69 P. asaccharolyticus 69 P. niger (Type species) 69 peptone 9, 30, 209 inhibition of growth by 209 peptone water 192 sugars (PWS) 30, 105, 198, 224 Peptostreptococcus 2, 51, 59, 68, 69, 176 P. anaerobius (Type species) 69 percentage results xvii, 180, 250 Petri dishes 191 pH indicators 12, 188 pH 9.6, growth at 66 phage types 1, 56, 70, 92, 114, 140 phase 141,265 phenolphthalein 189, 203 phenolphthalein phosphate medium 206, 234 phenol red 189, 224 phenon 265 phenotype 54, 265 phenylalanine medium 205, 206 test 39, 40, 234 phosphatase test 40, 56, 234, 235, 241 phosphate

buffer 222 effect on test 28 Photobacterium 121, 122; see Vibrio phthalate buffer 222 phylogenetic 173, 260, 265 pigmentation 21, 40, 97, 235 media for 197, 198 plague bacillus; see Yersinia pestis Planococcus 52 plasma for coagulase test 10, 31 plasmid xviii, 87, 265 (Plectridium tetani), 84 see Clostridium tetani Plesiomonas 2, 95, 121, 123, 127, 128, 176 P. shigelloides (Type species) 121, 123, 127, 128, 145, 267 Plimmer's mordant 216, 218 pneumococcus; see Streptococcus pneumoniae poly-6-hydroxybutyrate (PHB) 114, 235, 267 poly-6-hydroxybutyric acid (PHBA) 40, 206, 235, 267 polyclave 172, 173 polymorphism 24 polysaccharide antigens 62 porphyrin test 235 potato slopes 197 PPA (phenylpyruvic acid) test; see phenylalanine test PPLO, 16, 19, 267; see Mycoplasma Pragia 2, 149 P.fontium (Type species) 149 PRAS (pre-reduced) media 7, 8, 267 presumptive identification 19, 20, 33, 42, 56 Prevotella, 97; see Bacteroides primary tests 24-8, 48 principal components analysis; see numerical taxonomy probabilistic identification 48, 181 probe, DNA 1, 44, 123, 173, 265 probing punched cards 243, 244, 248 proficiency, laboratory 184, 186 profile numbers 182, 183 progressive method of identification 48, 49 pronunciation 169 Propionibacterium 2, 51, 75, 79, 81, 82 P. acnes %\,S2 P.avidumS\,S2 P. freudenreichii (Type species) 82 P. granulosumSl, 82 P. lymphophilum 82 Proteus 2, 5, 95, 128, 130, 136, 139 (P.hauseri) 139 P.mirabilis 136, 139 P.penned 136, 139 P. vulgaris (Type species) 136, 142, 239 Proteus OX strains 140 protozoa 15 Providencia 2, 5, 95, 128, 130, 139, 140 P. alcalifaciens (Type species) 139, 140, 142, 267 P. rettgeri 139, 140, 142, 240 P. rustigianii 140 P.stuartii 139, 140, 142 pseudocatalase 27, 75 Pseudomonas 2, 95, 109-11, 113-6 alkali producers 108-11 oxidizing pseudomonads 109, 113-6 P. acidovorans 108, 109 P. aeruginosa (Type species) 111, 113-6, 239 P. alcaligenes 108, 109

327

INDEX Pseudomonas (cont.) P.cepacia 111, 113, 115 P.diminuta 108, 109, 115, 116 P.fluorescens 111, 113, 115 P. mallei 111, 113, 114 P.maltophilia3\, 111, 115 P. mendocina 115, 116 P.paucimobilis 111, 115 P.pickettii 111, 115 P'. pseudoalcaligenes 108, 109 P. pseudomallei 111,113-5 P.putida 111, 115 P.stutzeri 111, 115, 116 P. testosteroni 108, 109 />. vesicularis 111, 116 psychrophilic organisms 87, 92, 93, 113, 265 pteridine reagent (O/129) 24, 39, 233 PTFE (polytetrafluoroethylene) filters 14 publication 170, 252, 253, 255 punched cards 47, 175-8, 243-8 pure cultures; purity checks xvii, 17-9, 48, 185 PWS; see peptone water sugars pyocyanin, medium for 113 pyruvate test 206, 236 qualitative and quantitative results 21, 22 quality assessment xix, 7 computer-assisted identification 180 external assessment 186 laboratory proficiency 186 QAL, Quality Assurance Laboratory 186, 267 quality control of media xix, 7, 184, 208 Rahnella 2, 149 R. aquatilis (Type species) 149 rank 1,168, 253, 255, 265 rapid methods 20, 42,44, 49 reagents for biochemical tests 219-22 reagents for staining procedures 214-6 record keeping 185 red mouth disease of trout, 146; see Yersinia rucked redox indicators 7, 12 resistance antiseptic 25, 114 heat 12, 18,25,41,66 (Rettgerella), 139, 140, 142; see Providencia rettgeri Rhodes' flagella stain 218 Rhodes' mordant 216,218 Rhodococcus 70,91 Rickettsia, 140; see Proteus R. mooseri 140 R. orientalis 140 R. prowazekii 140 Robertson's cooked meat medium 8, 186, 192 Rosenheim's test for tryptophan 209 Rothia 2, 51,19, S2 R. dentocariosa (Type species) 81, 82 Rules of nomenclature 5, 6, 170, 252-3 safety, laboratory xvii, 187 safranin 214 saline agar 194 Salmonella 2, 95, 128, 130, 140-2 S. arizonae 141, 142

328

S. choleraesuis (Type species) 141-3 S. gallinarum 140-2 (S.houtenae) 141, 142 (S.kaufmannii) 141, 142 S.paratyphiA 140-3 S.pullorum 140-2 (S.salamae) 141, 142 S. typhi 140, 239 (5. typhimurium) 239 salmonella subgenera 141 Salmonella subgenus I; S. enteritidis; (S. kaufmannii) 141, 142 Salmonella subgenus II; (S. salamae) 141, 142 Salmonella subgenus III; S. arizonae 141, 142 Salmonella subgenus IV; (S. houtenae) 141, 142 salt salt colistin broth 197 other salt media 104, 207 salt requirement 122, 236 sarcinas 53 Scotochromogenic psychrophils; see Mycobacterium screening of cultures 19, 48 screw-capped bottles 16, 17, 20, 30, 188, 189 secondary tests 2 8 ^ 2 , 48, 49 selenite media 141, 207, 236 semisolid media motility testing, for 191, 232 sensitivity antibiotic xviii, 9, 17, 23, 24, 36, 43, 44, 69 bacteriocin 17, 114 bacteriophage 1, 56, 70, 92 to dyes 105, 118 test 27, 36, 49 serology xvii, 23, 54 serotypes xviii, 1, 64, 66, 140, 169, 177, 264 serovars 82 Serratia 2, 95, 128, 130, 143-5 S.ficaria 145 (S.fonticola) 145 S. liquefaciens 143, 144 S. marcescens (Type species) 143-5, 239, 240 S. marinorubra 143 S. odorif era 143 S.plymuthica 144, 145 (S. rubidaea); see S. marinorubra serum 10 amylase in 10 anticomplementary 9 digestion 33, 228 maltase in 10 media 195, 198,201,224 shelf life of media 185 Shewanella2,95,lW-9,122 S. putrefaciens (Type species) 108, 109 Shiga's bacillus, 95; see Shigella dysenteriae 1 Shigella 2, 95, 128, 130, 144-6 S. boydii 144, 145 S. dysenteriae (Type species) 95, 144-6, 239 S.flexneri 144-6 S. sonnei 144,146,239 signs and symbols used in tables 47 similarity coefficients 168, 265 similarity matrix; see numerical taxonomy similarity of characters 21 Simmons' citrate medium 31, 201

INDEX Smegmatis-phlei group; see Mycobacterium smell 22, 73, 107, 113 soil extract medium 207 spore-forming stimulant 25 S0rensen's citrate buffer 222, 235 species 1, 4, 167, 252, 253, 255, 258 exotic 48 indefinable nature of 1 new 5, 48, 252 subdivisions 1, 253 specimens, bacteriological examination of culture 15-9 microscopy 15 (Sphingobacterium) 116 (S. multivorum) 117, 267 (S. spiritovorum) 117 spirochaetes 4, 15, 24, 161 spores 25 position 85, 97 resistance 25, 41, 85 shape 85, 87 stain 25, 217 Sporolactobacillus 11 Sporosarcina 52 'spreaders' 18; see also swarming sputum 15, 45, 92 SQL computer language 249, 267 stains capsule 217 differential 216 flagella24,215, 218 reagents 214—8 simple 214, 216 special 217 spore 25, 217 Staphylococcus 2, 51-7 biotypes 54, 56 coagulase 31, 54, 56, 226 haemolysins 54, 56 leucocidins 56 phage typing 56 protein A 56 serology 52 toxins 54, 56 S. ariettae 55 S. aureus (Type species) 54-7, 239 S. auricularis 55 S. capitis 54—6 S. caprae 55, 56 S. carnosus 55 S. caseolyticus 53, 55 S. chromogenes 55, 56 S. cohnii 55, 57 S. epidermidis 53-7 iS. equorum 55 S. gallinarum 55, 57 S. haemolyticus 54-6 S. hominis 54-6 S. hyicus 53, 55, 56 S. intermedius 53, 55, 56 S. kloosii 55 S. lentus 55, 57 S. lugdunensis 54, 55

S. saccharolyticus 55, 56 S. saprophyticus 53-7, 239 S. schleiferi 55 S. sciuri 53, 55-7 S. simulans 53, 55, 56 S. warned 55, 56 S. xylosus 55, 57 starch 211 agar 200 hydrolysis 236 statistical methods 167, 181 steam relation between temperature and pressure 13 sterilization by 14 sterility, testing 25, 212 sterilization xvii, 12 autoclaving 12, 14 carbohydrate 11, 198-200 control 87, 212 discarded cultures 12, 187 filtration 14 fractional 14 glassware 188 hot air 13 inspissation 14 steaming 12 Tyndallization 14 stock control of media 185 Stomatococcus mucilaginosus (Type species) 2, 51-3, 58 Streptobacillus 2,95, 150, 151, 161, 176 S. moniliformis (Type species) 151, 161 Streptobacterium, 75; see Lactobacillus streptococcal extracts for grouping 236 Streptococcus 2, 51, 59-66, 176 faecal streptococci, 66, 67; see Enterococcus group D streptococci 62, 65, 66; see Enterococcus grouping, Lancefield 62, 64-6 haemolysis 62 motile strains 67 S. agalactiae 29, 60, 64, 239 S. anginosus 60, 63, 64, 239 S. bovis 6\, 65, 66, 6S S. canis 60, 63, 64 S. constellatus 60, 64 S. cricetus 66 S. dysgalactiae 60, 63, 239 S. equi 60, 63 S. equinus 61, 65, 66 S. equisimilis 60, 63 S. intermedius 64 (S. milled) 63, 64, 239 (S. mitior) 64, 65 S. morbillorum 60, 65, 68 (S. mundtii) 61; see Enterococcus mundtii S. mutans 61, 65, 66 S. oralis 60, 64, 65 S. pneumoniae 29, 60, 239 S. porcinus 60, 64 S. pyogenes (Type species) 60, 63, 64, 66 S. rattus 66 S. salivarius 61, 62, 66, 239 S. sanguis 60, 65, 239 S. sobdnus 66 S. suis 60, 64

329

INDEX Streptococcus (cont.) S. uberis 60, 64 S. zooepidemicus 60, 63 Streptomyces 46, 79, 172, 207 'string of pearls' test 237 Stuart's urea medium 208, 238 subspecies 1, 252, 255, 265 sugar, media containing 19, 27, 30, 198-200 ASS (ammonium salt sugars) 31, 115, 198, 266 for dextran and levan production 60, 61, 68, 235 for lactobacilli 199 for neisserias 199 PWS (peptone water sugars) 30, 198, 224 sugar reactions 27, 30 survival of bacteria 25, 40 swarming of cultures 5, 18, 84, 85, 140, 191 prevention of 18, 140 symbiosis 265 symbols used in tables 47 synergy, -ism 17, 98, 265 synonym xviii, 1, 5, 6, 68, 253, 265 systematics; see numerical taxonomy tables of characters xviii, 46-9 development xvii, 47 Gram-negative bacteria 94—164 Gram-positive bacteria 50—93 (Tatlockia), 163; see Legionella Tatumella 2,95, 128, 146 T. ptyseos (Type species) 130, 144, 146 taxon 1,4,265 taxonomy ix, xiii, 1, 3, 165-74, 261 taxon-radius model; see identification statistics TCBS medium 122, 197 Teepol containing media 122, 196 tellurite media 196 tolerance 237 temperature effect on isolation 141 effect on tests, 26, 28, 33, 36, 40, 41, 137, 147, 148 optimal 41 rangeofgrowth41,73,87,92, 112, 113, 121, 126, 237 sterilization 12-4 tolerance 41, 66, 237, 238 Terrae group; see Mycobacterium test error 22, 168, 180 test organisms 239^-2 tests characterization 20, 219-38 primary 24-8 rapid 20, 42, 44, 49 screening 19,20,48 secondary 28^12 selection 180 tetrazolium 12,26 Thermobacterium, 75; see Lactobacillus thermophil(e) 87, 92, 212, 266 thioglycollate medium 193, 210 Todd-Hewitt broth 207 tomato juice medium 195 toxigenicity testing 123, 241 toxins 35, 54, 56, 71, 72, 83, 86, 125 transformation, coccal 160

330

transport medium 13, 17 Triple sugar iron (TSI) medium 9, 207, 267 tryptophan 43, 209 tubercle bacilli; see Mycobacterium tuberculosis Tween80,92, 156, 157 medium 208 mixture 221 test 41, 238 Tyndallization 14 type concept 258 nomenclatural 170, 258, 266 strain xiii, 170, 253, 258, 266 tyrosine agar 208 decomposition 227 unexpected organisms 15 urea, media containing 208 Ureaplasma urealyticus 164 urease, production of 41, 238 urethral smears 15 urinary tract infection (UTI) 54, 57, 82 utilization acetylmethylcarbinol 28, 222 citrate 31, 201 indole 36 malonate 231 organic acids 31, 201, 202 ' V factor 150-6, 222, 238, 241 valid publication 252, 266 variable character 23, 86, 167 Gram reaction 3, 4, 86, 100 variant 65, 177,266 aerogenic 135, 145 anaerogenic 135, 141, 177 asporogenous 49 geographic 92, 104, 177, 180 motile and non-motile 49, 75, 177 variety (of species) 1, 70, 88, 89, 266 Veillonella 2, 94-9 V. atypica 98 V. dispar 98 V. parvula (Type species) 98 Vibrio 2,95, 121-7, 176 NAG; non-agglutinable vibrio 125, 267 NCV; non-cholera vibrio 125, 267 V. alginolyticus 122-6, 239 V. anguillarum 1 2 2 ^ , 126 V. cholerae (Type species) 122-6 V. cincinnatiensis 123, 124, 126

V.damsela 123, 124, 126 V.fluvialis 121-4, 126, 127, 267 V.furnissii 122-4, 127,239 V. harveyi \23, 124, 126 V.hollisae 122-4, 126 V. metschnikovii 122^, 126 V. mimicus 122-5 V. natriegens 123, 124, 126 V. parahaemolyticus 122-6, 239 V. proteolyticus 126 V. vulnificus 123, 124, 126

INDEX

Vibrionaceae 122 Vincent's angina 97 violacein 128 'violagabriellae' 54 Voges-Proskauer (VP) test 37, 222, 267 media for 199, 200 volumes of media 191 volutin, stains for 217 water 192 water agar 31, 192, 194 Weeksella 2, 95, 109, 116, 117 W. virosa (Type species) 117, 267 W.zoohelcum 117,267 weighting of characters 21 whooping cough 106 (Wolinella) 160 (W. succinogenes) (Type species) 160 'X' factor 150-6, 222, 238, 241 xanthine agar 208 decomposition 227 Xanthomonas 111

Xenorhabdus 2, 149 X. luminescens 149 X. nematophilus 149 yeast 10,21 yeast extract 10, 194, 209 Yersinia 2, 95, 119, 121, 128, 130, 144, 146-8 Y. aldovae 146-8 Y. bercovieri 146, 147, 267 Y. enterocolitica 118, 119, 144, 146-8, 267 Y.frederiksenii 144, 146-8 Y. intermedia 144, 146-8 Y. kristensenii 144,146-8 Y. mollaretii 146, 147, 267 Y. pestis (Type species) 27, 118, 119, 121, 144, 146-8 (Y. philomiragia) 121, 147; see Francisella Y.pseudotuberculosis 118, 119, 144, 147, 148 Y. rohdei 146, 147 Y. ruckeri 146, 147 Yokenella 149 Y. regensburgei (Type species) 149; see Koserella trabulsii Ziehl-Neelsen staining method 15, 25, 92, 217 zinc dust 38, 39, 232

331